MA3-915

antibody from Invitrogen Antibodies

Targeting: ATP1A3 DYT12

Provider product page for MA3-915

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [99]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [4]
Immunohistochemistry [3]
Other assay [42]

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Product number: MA3-915 - Provider product page
Provider: Invitrogen Antibodies
Product name: ATP1A3 Monoclonal Antibody (XVIF9-G10)
Antibody type: Monoclonal
Antigen: Other
Description: MA3-915 detects sodium/potassium ATPase from human, monkey, bovine, sheep, canine, rabbit, guinea pig, mouse and rat tissue. This antibody is specific for the alpha-3 subunit.
Antibody clone number: XVIF9-G10
Concentration: 1 mg/mL

ATP1A3 protein structure - MA3-915 shown in red.

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Experimental details: Knockdown of ATP1A3 was achieved by transfecting SH-SY5Y with ATP1A3 specific siRNAs (Silencer® select Product # s1724 and s1725). Western blot analysis (Fig. a) was performed using Whole cell extracts from the ATP1A3 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ATP1A3 Monoclonal Antibody (XVIF9-G10) (Product # MA3-915, 0.25 µg/ml) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to ATP1A3. An uncharacterized band (*) was observed at around 80 kDa.

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Experimental details: Western blot was performed using Anti-ATP1A3 Monoclonal Antibody (XVIF9-G10) (Product # MA3-915) and a 110 kDa band corresponding to ATP1A3 was observed across cell lines and tissue tested. Whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1), IMR-32 (Lane 2), SK-O-V3 (Lane 3), HeLa (Lane 4) and Mouse Brain (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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Experimental details: Immunofluorescent analysis of Sodiµm/Potassium ATPase alpha-3 using Sodiµm/Potassium ATPase alpha-3 Monoclonal antibody (XVIF9-G10) (Product # MA3-915) shows staining in C6 glioma cells. Sodiµm/Potassium ATPase alpha-3 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Sodiµm/Potassium ATPase alpha-3 (Product # MA3-915) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of Sodiµm/Potassium ATPase alpha-3 using Sodiµm/Potassium ATPase alpha-3 Monoclonal antibody (XVIF9-G10) (Product # MA3-915) shows staining in HeLa cells. Sodiµm/Potassium ATPase alpha-3 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Sodiµm/Potassium ATPase alpha-3 (Product # MA3-915) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of Sodiµm/Potassium ATPase alpha-3 using Sodiµm/Potassium ATPase alpha-3 Monoclonal antibody (XVIF9-G10) (Product # MA3-915) shows staining in U251 glioma cells. Sodiµm/Potassium ATPase alpha-3 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Sodiµm/Potassium ATPase alpha-3 (Product # MA3-915) at a dilution of 1:20 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescence analysis of ATP1A3 was performed using SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with ATP1A3 Monoclonal Antibody (XVIF9-G10) (Product # MA3-915) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human colon tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Sodiµm/Potassium ATPase alpha-3 (Product # MA3-915) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human tonsil tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing Sodiµm/Potassium ATPase alpha-3 (Product # MA3-915) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human prostate carcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a mouse monoclonal antibody recognizing Sodiµm/Potassium ATPase alpha-3 (Product # MA3-915) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Figure 2 In vivo expression of mutant Atp1a2 . Total RNA and protein samples were isolated from brain of wild type (+/+), Atp1a2 +/R887 (+/R887) and homozygous Atp1a2 R887/R887 (R887/R887) mice at E19.5. A. Left panel. Semi quantitative Atp1a2 RT-PCR (254 bp fragment) on brain cDNA. The b-actin fragment (610 bp) is included as in-tube normalizer. Right panel, same Atp1a2 RT-PCR fragments digested by MspI. B. Protein blot of microsomal fraction probed with anti-alpha2 Na,K-ATPase antibody and anti-neogenin as loading control; the alpha2 Na,K-ATPase and neogenin bands appear at the expected size of 110 kDa and 52 kDa, respectively. C. Total brain lysates from adult wild type and Atp1a2 +/R887 mice probed with anti-alpha1, alpha2, and alpha3 Na,K-ATPase antibodies; anti- tubulin as loading control. Densitometric quantization shows a 50% reduction of the heterozygous mutant alpha2 level compared to wild type. Error bars represent +- SD; Student's t test p

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Experimental details: Figure 3 In vitro expression and localization of alpha2-ATPase . A. Immunoblot of HeLa cells transfected with pA2-wt-Myc (wt) and pA2-R887-Myc (R887) probed with anti alpha2-ATPase antibody. B. Calnexin (red) and alpha2 ATPase (probed with anti-myc; green) immunofluorescence staining of wt or R887 transfected Hela cells. R887 alpha2 ATPase mostly colocalises with the endoplasmic reticulum. In the right graphs, the scatter plot of red (ch1) vs. green (ch2) colocalization intensities. (wt parameters: Rr 0.369, R 0.6, Ch1:Ch2 0.999; R887 parameters: Rr 0.558, R 0.755, Ch1:Ch2 0.997). C. HeLa cells expressing wt or R887 variant of alpha2 ATPase are treated with the reversible proteasome inhibitor MG132 (10 uM for 4, 6 and 8 hrs). Blots are probed with anti- alpha2 isoform antibody, anti-ubiquitin antibody and anti-tubulin as loading control.

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Experimental details: Figure 4 Tonotopic distribution of BK channels in outer hair cells. 3D renderings of confocal z-stacks of apical (A), middle (B), and basal (C) cochlear turns immunostained with a monoclonal antibody against the BK channel (red) and a polyclonal antibody against synapsin (green) to label the efferent presynaptic terminals show that BK channel immunoreactivity in the three rows of outer hair cells (when present) is associated with efferent terminals. (D) Moreover, the size of BK channel immunopuncta increases from apical to middle and basal turns, paralleling an increase in size and likely number of efferent terminals. Data are presented as box plots representing the median (box interior), the 25th and 75th percentile (box boundaries), the 10th and 90th percentile (whiskers), and the 5th and 95th percentile (dots). (E) 3D renderings of confocal z-stacks of a middle cochlear turn immunostained with a polyclonal antibody against the BK channel (red) and a monoclonal antibody against the NaK-ATPase alpha3 (green) to label the efferent presynaptic terminal membrane shows adjacent but not co localized expression of the BK channel with the presynaptic efferent terminal membrane. The location of the three rows of outer hair cells are indicated (solid arrowheads). Grid dimensions equal 10 um.

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Experimental details: Figure 1 Co-immunoprecipitation and GST pull down assay of PSD-95 and alpha3 NKA . Co-immunoprecipitation (CoIP) shows interaction between PSD-95 and alpha3 NKA. GST pull down assays shows interaction of PSD-95 with the alpha3 NKA N-terminus and a specific PDZ3 interaction.

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Experimental details: Figure 3 STED microscopy dissecting the localization of Na + ,K + -ATPase in cultured striatal neurons . The 5 x 3 mosaic shows a comparision of the confocal (left) and STED images (middle) of the Alexa-594 immunolabelled alpha3 NKA in dendritic spines. Postprocessing of the raw STED data by a Richardson-Lucy deconvolution further enhances the details as shown in the right row of images. Scale bar: 500 nm

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Experimental details: Figure 2. alpha-actinin-1 and alpha9/10-nAChRs interact with SK2 channels in Xenopus oocytes. Immunoblots show co-precipitation of exogenous SK2 with co-expressed alpha-actinin-1 ( A ) and HA-tagged alpha9/10-nAChRs ( B ) in Xenopus laevis oocytes. As negative controls, alpha-actinin-1 and nAChRs did not co-precipitate with an unrelated antibody of the same IgG subclass (anti-mGluR5; ctl lane in A and B ) and SK2 did not co-precipitate with endogenous Na + /K + ATPase ( B , lower panel). Input, 6% of total membrane fraction (M) and lysate (tot) for all panels. n = 4 separate experiments.

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Experimental details: Figure 5 Comparison of protein expression in LV sample from control and cardiomyopathic patients. A: Top; representative SERCA2 and respective GAPDH stainings obtained in normal and cardiomyopathic LV samples. Bottom; Mean+-SEM, *** P

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Experimental details: Figure 4 Representative western blot images for each NKA isoform analyzed ( alpha 1-3 and beta 1-2 ), including example of the coomassie stain used to normalize blot density to the total protein content of the sample. ""Inj"" represents the injured leg from the KI group, ""Non"" represents the noninjured leg from the KI group, and ""CON"" represents the control group.

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Experimental details: Fig. 1 Differential trajectories of the cochlear afferent fibers (CAFs) in the organ of Corti. (A) A three-dimensional (3D) reconstructed image of the organ of Corti immunolabeled with anti-calretinin in a P12 rat. (Aa~Ab) XY and ZY projection views of the marked area in A. (B) 3D-reconstructed images of an organ of Corti in a P11 rat that is double-immunolabeled with anti-calretinin (green) and anti-neurofilament (NF; red). (Ba1~Ba3) ZY section views of the marked area in B. An approximate location of habenular perforata is marked with dotted oval. (C) A 3D reconstructed image of an organ of Corti from a P19 rat that was double-immunolabeled with anti-calretinin (green) and anti-NF (red). (Ca1~Ca3) ZY projection views of the marked area in C. (D) A 3D reconstructed image of an organ of Corti from a P19 rat that was triple-immunolabeled with anti-calretinin (green), anti-myosin (red) and anti-NKA (magenta). (Da1~Da5) ZY section views of the marked area in D. (E) A 3D reconstructed image of an organ of Corti from a 11-week-old rat (W11) that was double-immunolabeled with anti-calretinin (green) and anti-Na, K-ATPase alpha 3 (NKA; red). (Ea1~Ea3) ZY projection view of the marked area in E. At all ages examined, calretinin-rich and calretinin-poor fibers (arrow) exhibit segregated courses. The pillar (P) and modiolar side (M) of IHC is marked with dotted lines (Ab, Ba3, Ca3, Da5, and Ea3).

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Experimental details: FIGURE 7 Subcellular fractionation by density gradient ultracentrifugation of human putamen. Dorsal putamen was fractionated as described in section ""Materials and Methods."" Fractions were assessed by SDS-PAGE and western blot analysis using different cell compartment markers. Fractions 1-16 were removed from top to bottom. Equal volumes of each fraction were loaded per lane. (A) Diagram at left shows approximate distribution of proteins representative of different subcellular compartments in the control putamen. (B) Shown are representative images of western blots detecting GLUT3 and SNAP25 in control and HD putamen. (C) Shown are WB images of Huntingtin (HTT) and mutant HTT (mHTT) signals in 16 fractions from all human putamen samples. mHTT was detected with 1C2 which recognizes the expanded polyQ region in mutant HTT but not HTT in control samples. (D) Bar graph shows mean percent +- SD of HTT signal obtained with antibody Ab1 in control and HD human putamen in each of 16 fractions as a percent of total HTT signal summed across all fractions. There was no significant difference in HTT signal between control and HD in any of the fractions ( N = 3). (E) Fractions 3-8 were analyzed in pairs and run on the same gel to eliminate variability due to different runs. These fractions were chosen because most proteins tested had peaks in this range. Seven probes were analyzed by western blot for each gel (Calnexin, GLUT3, Na+/K+ ATPase, NMDAR 2b, PSD95, SNAP25, and XK). Band intens

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Experimental details: Fig. 1 PRRT2 interacts with alpha3-NKA and alpha1-NKA subunits. A Pulldown of alpha3-NKA and alpha1-NKA by PRRT2 : Affinity-purified PRRT2-HA or BAP-HA were incubated with total mouse brain extracts. After pulldown (PD), pellets were solubilized and subjected to western blotting with alpha3-NKA and alpha1-NKA antibodies. Mouse brain lysates incubated with PRRT2-HA showed specific immunoreactivity for alpha3-NKA and alpha1-NKA in the precipitates, which were not detected with BAP-HA. Top: Representative immunoblots are shown. Bottom: Quantification of the alpha3-NKA and alpha1-NKA immunoreactivities in the pulled down samples normalized to the BAP-HA control (means +- SEM, n = 4 independent experiments, ** p < 0.01; unpaired Student's t -test). Input, 10 mug total extract. B Co-immunoprecipitation of PRRT2 with alpha3-NKA and alpha1-NKA antibodies : Detergent extracts of mouse brain were immunoprecipitated (IP) with monoclonal antibodies (mAbs) to alpha3-NKA, alpha1-NKA or GFP (used as a control), as indicated. After the electrophoretic separation of the immunocomplexes and western blotting, membranes were probed with alpha3-NKA or alpha1-NKA antibodies to test the immunoprecipitation efficiency, as well as with polyclonal PRRT2 antibodies to test for co-immunoprecipitation. Left: Representative immunoblots are shown. Right: Quantification of the PRRT2 immunoreactive signal in the immunoprecipitated samples normalized to the GFP nonspecific signal (means +- SEM, n = 4 independ

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Experimental details: Fig. 2 Co-localization of alpha3-NKA and alpha1-NKA with PRRT2 in primary hippocampal neurons. A , B Hippocampal neurons (DIV 14) were stained for endogenous PRRT2 and either alpha3-NKA ( A ) or alpha1-NKA ( B ). Left: Representative confocal images show the overlapping staining of NKA isoforms and PRRT2 (magnified in the insets). Scale bar, 10 mum. Right: Quantitative evaluation of the extent of the codistribution between PRRT2 and NKA isoforms using the Manders' correlation coefficient expressing the PRRT2/NAK overlap area in percent of the NAK immunoreactive area (0.96 +- 0.06 and 0.87 +- 0.04 for alpha3-NKA/PRRT2 and alpha1-NKA/PRRT2, respectively; n = 15 images from three independent experiments). C , D Hippocampal neurons transfected with PRRT2-HA and either alpha3-NKA-SEP ( C ) or alpha1-NKA-SEP ( D ) were live-labeled (DIV 14) with anti-HA and anti-GFP antibodies to stain the surface-exposed domains of the proteins. Left : The images show the largely overlapping staining of PRRT2 with both alpha3-NKA-SEP and alpha1-NKA-SEP (magnified in the insets). Scale bar, 10 mum. Right: Quantitative evaluation of the extent of the codistribution between PRRT2-HA and the NKA-SEP isoforms using the Manders' correlation coefficient expressing the PRRT2/NAK overlap area in percent of the area of NAK fluorescence (0.91 +- 0.04 and 0.84 +- 0.03 for alpha3-NKA/PRRT2 and alpha1-NKA/PRRT2, respectively; n = 15 images from three independent experiments).

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Experimental details: Fig. 3 PRRT2 does not affect NKA expression level at the membrane surface. A , B Left: Representative immunoblots of cell surface biotinylation performed in WT and PRRT2 KO hippocampal neurons. Total lysates (TOTAL), biotinylated (cell surface, EXTRA), and non-biotinylated (intracellular, INTRA) fractions were analyzed by western blotting with antibodies to PRRT2 and either alpha3-NKA ( A ) or alpha1-NKA ( B ). Antibodies to transferrin receptor (Transf Rec) and actin were used as markers of cell surface and intracellular fractions, respectively. Vertical lines in the blot indicate that the lanes were on the same gel but have been repositioned in the figure. Right: Total and cell surface alpha3-NKA ( A ) and alpha1-NKA ( B ) immunoreactivities are expressed in percent of the respective WT value after normalization to Transferrin receptor (for the EXTRA fraction) or actin (for the INTRA fraction). Means +- SEM of n = 3 independent experiments; unpaired Mann-Whitney's U -test. C , D Left : Representative images of a WT neuron transfected with alpha3-NKA-SEP ( C ) or alpha1-NKA-SEP ( D ) perfused with Tyrode followed by MES buffers (scale bar, 20 mum). Insets show higher magnification of the fluorescence at the plasma membrane where ROIs were measured. The cartoons shown on top depict the SEP state under Tyrode and MES buffers, respectively. The green stars represent fluorescent emission of the chimeric protein, which is quenched at acidic pH, i.e., in acidic intracellular compa

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Experimental details: Fig. 4 PRRT2 affects alpha3-NKA clustering at the membrane surface. A , B Representative 3D-SIM ( A ) and STED ( B ) images of hippocampal neurons (DIV 14-17) stained for alpha3-NKA. The images highlight an altered clustering of alpha3-NKA along the plasma membrane in PRRT2 KO neurons as compared to WT neurons that is rescued by expression of PRRT2 in PRRT2 KO neurons (KO + PRRT2). Scale bar, 5 mum; insets: 1 mum. C , D Size distribution of alpha3-NKA membrane clusters. Clusters were subdivided into bins on the basis of their size as indicated. The histograms (means +- SEM) show the area occupied by each bin of alpha3-NKA clusters expressed in percent of the total immunoreactive area. Both 3D-SIM ( C ) and STED ( D ) microscopy showed a significant increase in the area of largest clusters and a reciprocal decrease in the area of smallest clusters along the soma and the dendritic membrane in PRRT2 KO neurons (red bars) with respect to WT neurons (black bars). The change was fully rescued by re-expression of PRRT2 in PRRT2 KO neurons (KO + PRRT2, blue bars). Data refer to n = 35 (3D-SIM) and 25 (STED) neurons per genotype, from n = 3 independent preparations (3 coverslips/preparation/genotype). ** p < 0.01, *** p < 0.001; one-way ANOVA/Bonferroni tests.

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Experimental details: 10.1371/journal.pone.0258682.g004 Fig 4 Validation of NKA binding to PrP. (A) Western blot-based validation of co-immunoprecipitation of NKA subunits with PrP. (B) Evidence of partial cellular co-localization of PrP C and ATP1A1 by co-immunocytochemical analysis of ReN VM cells, with Hoechst stain serving as the nuclear counter stain. Scale bar: 10 nm. (C) Chemical structure of the NKA inhibitor ouabain. (D) Parallel ouabain concentration-dependent effects on NKA and PrP C levels. Exposure of differentiated ReN VM cells to ouabain at concentrations up to 50 nM causes a biphasic effect on PrP C and ATP1A1 levels, with concentrations up to 30 nM leading to a ouabain concentration-dependent reduction in steady-state levels of both proteins, and exposure to 50 nM provoking a reproducible slowing of PrP C during SDS-PAGE separation that parallels an increase in steady-state ATP1A1 levels.

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Experimental details: 10.1371/journal.pone.0258682.g008 Fig 8 The steady-state levels of isoforms of PrP C and NKA alpha-subunits are individually controlled by extracellular CGs and intracellular Ca 2+ ions. Exposure to 20 nM ouabain caused a reduction in the steady-state levels of PrP C , ATP1A1 and ATP1A2--but not ATP1A3--in a manner that cannot be reversed by concomitant incubation with YM24476. In contrast, co-incubation of cells with ouabain and A6185 rescued the reduction in ATP1A1 levels and caused the appearance of signals detected with the PrP C - and ATP1A2-directed antibodies that migrated with different apparent molecular weights than the dominant signals for these proteins observed in untreated cells. Finally, the intensity of relatively fast migrating signals detected with a GFAP-directed antibody increase under conditions that favor intracellular calcium-dependent calpain activity.

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Experimental details: Figure 1. Na,K-ATPase isoforms are reduced in gene-targeted mice. A , Reduced Na,K-ATPase alpha3 isoform mRNA expression in embryonic E18.5 d brains from WT, heterozygous (+/-), and homozygous (-/-) alpha3 knock-out mice by Northern blot. L32 was used as a loading control. Twenty micrograms of total RNA were loaded per lane. B , RT-PCR analysis of RNA from WT, heterozygous (+/-), and homozygous (-/-) alpha3 knock-out mice shows larger RNA transcript in -/- (arrow) but not +/- or WT mice. C , Western blot analysis of Na,K-ATPase isoforms in whole tissue extracts from hippocampus of adult male alpha1 +/- , alpha2 +/- , and alpha3 +/- mice. Total protein loaded per lane was as follows: 10, 0.5, and 1 mug for alpha1, alpha2, and alpha3 isoform expression, respectively. D , Semiquantitation by densitometry on whole tissue extracts from adult hippocampus of alpha1 +/- , alpha2 +/- , and alpha3 +/- mice shows reduction in alpha1, alpha2, and alpha3 isoforms, respectively. * p < 0.05 versus WT.

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Experimental details: Fig 2 Retinoschisin binding in dependence of ATP1B2 glycosylation. (A) HEK293 cells were co-transfected with expression constructs for ATP1A3 and expression constructs for ATP1B2 mutants deficient in each one glycosylation site (ATP1B2-N96Q, -N118Q, -N153Q, -N159Q, -N193Q, -N197Q, -N238Q, and -N250Q). 48 h after transfection, cells were incubated with recombinant retinoschisin for 1 h, followed by intensive washing. Cells transfected with only ATP1A3 expression constructs served as a negative control, cells co-transfected with expression constructs for ATP1A3 and for normal ATP1B2 served as a positive control in the retinoschisin binding assay [ 13 ]. Heterologous protein expression as well as retinoschisin binding was investigated by Western blot analyses with antibodies against retinoschisin, ATP1A3, and ATP1B2. The ACTB staining served as loading control. (B) Enriched membrane fractions from HEK293 cells transfected with ATP1A3 and ATP1B2 expression constructs and Y79 cells were subjected to enzymatic deglycosylation using PNGase F. A control sample was subjected to the same treatment, but without PNGase F. Subsequently, membrane fractions were incubated with recombinant retinoschisin for 1 h, followed by intensive washing. Retinoschisin binding, (heterologous) Na/K-ATPase expression, and ATP1B2 deglycosylation were investigated by Western blot analyses with antibodies against retinoschisin, ATP1A3, and ATP1B2. Membranes from HEK293 cells transfected with only ATP1A3 e

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Experimental details: Fig 4 Retinoschisin binding to ATP1B2 patch I mutants. HEK293 cells were transfected with expression constructs for ATP1A3 in combination with expression constructs for different ATP1B2 variants mutated in patch I regions depicted within the figure. (A) 48 h after transfection, cells were subjected to recombinant retinoschisin for 1 h, followed by intensive washing. Cells transfected with expression constructs for only ATP1B2 served as a negative control, cells transfected with expression constructs for ATP1A3 and normal ATP1B2 served as a positive control in the retinoschisin binding assay [ 13 ]. Heterologous protein expression as well as retinoschisin binding was investigated by Western blot analyses with antibodies against retinoschisin, ATP1A3, and ATP1B2. The ACTB staining served as loading control. (B) 48 h after transfection, cell surface proteins were biotinylated, purified by streptavidin affinity chromatography and analysed by Western blotting using antibodies against ATP1B2. The ATP1A1 immunoblot was performed as loading control. (C) 48 h after transfection, cells were subjected to FACS analyses applying an anti-ATP1B2 antibody. Representative histograms of cells transfected with selected ATP1B2 patch I mutants are given in this figure. Light grey: histogram of untransfected HEK293 cells depicting unspecific background signals. Dark grey: histogram of transfected HEK293 cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 6 Retinoschisin binding to ATP1B2_T240A and ATP1B2_T240S. HEK293 cells were transfected with expression constructs for ATP1A3 in combination with expression constructs for ATP1B2_T240A and ATP1B2_T240S. (A) 48 h after transfection, cells were subjected to recombinant retinoschisin for 1 h, followed by intensive washing. Cells transfected with expression constructs for only ATP1B2 served as a negative control, cells transfected with expression constructs for ATP1A3 and normal ATP1B2 served as a positive control in the retinoschisin binding assay [ 13 ]. Heterologous protein expression as well as retinoschisin binding was investigated by Western blot analyses with antibodies against retinoschisin, ATP1A3, and ATP1B2. The ACTB staining served as loading control. (B) 48 h after transfection, cells were subjected to FACS analyses applying an anti-ATP1B2 antibody, representative histograms are given in this figure. Light grey: histogram of untransfected HEK293 cells depicting unspecific background signals. Dark grey: histogram of transfected HEK293 cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Muscle NKA alpha 1 (A), alpha 2 (B), and alpha 3 (C) isoform relative abundance from the vastus lateralis of the injured and noninjured legs of participants with knee injury (KI) and from a single leg of age- and BMI-matched controls (CON). Representative western blots are included; ""Inj"" represents the knee-injured leg, ""Non-I"" represents the noninjured leg, and ""CON"" represents the control group. Unfilled bars represent the injured leg from KI, filled bars represent the noninjured leg from KI and the hatched bars represent CON. *Less than noninjured leg ( P < 0.05), &ddagger; Moderate effect size compared to noninjured leg. Values are Mean +- SD, n = 6 for each leg KI, n = 7 CON.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Muscle NKA beta 1 (A) and beta 2 (B) isoform relative abundance from the vastus lateralis of the injured and noninjured legs of participants with knee injury (KI) and from a single leg of age- and BMI-matched controls (CON). Representative western blots are included; Inj represents the knee-injured leg, Non-I represents the noninjured leg, and CON represents the control group. Hollow bars represent the injured leg from KI, filled bars represent the noninjured leg from KI and the hatched bars represent the CON. &ddagger; Moderate effect size compared to noninjured leg in KI. Values are Mean +- SD, n = 6 for each leg KI, n = 7 CON.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 Validation of the identified proteins in dendritic filopodia. (A,B) Localization of the identified proteins in dendritic filopodia (A1-A21) and phagocytic cups (B1-B21) at 14 DIV hippocampal neurons. The cultured neurons were immunostained with anti-TLCN antibody and specific antibodies against Galphao (A1,B1) , Na + /K + ATPase alpha3 (A2,B2) , EFA6D (A3,B3 ), spectrin (A4,B4) , septin7 (A5,B5) , myosin VA (A6,B6) , Galphaq (A7,B7) , Gbeta2 (A8,B8) , eEF1gamma (A9,B9) , ribosomal protein S16 (A10,B10) , CaMKIIalpha (A11,B11) , CD98hc (A12,B12) , EFA6C (A13,B13) , BAIAP2L1 (A14,B14) , PLCbeta3 (A15,B15) , MRCKalpha (A16,B16) , NR3A/B (GluN3A/B) (A17,B17) , SAP97 (SLC3A2) (A18,B18) , MAP1S (A19,B19) , EPS8L1 (A20,B20) , and JIP-4 (SPAG9) (A21,B21) . In merged images, the identified proteins and TLCN are shown in magenta and green, respectively. Single plane of images focused on dendritic filopodia (A1-A21) and center of microbeads (B1-B21) were acquired using a confocal microscopy. Scale bars, 5 mum in (A14,A21,B14,B21) .