MA3-921

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Dihydropyridine Receptor alpha-2 was performed by loading 25 µg of U87-MG (lane 1) and mouse brain (lane 2) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~143 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using CACNA2D1 Monoclonal Antibody (20A) (Product # MA3-921) and a ~140 kDa band corresponding to CACNA2D1 was observed along with few uncharacterized bands (*) in the range of 80 kDa – 15 kDa. Whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1) A549 (Lane 2), PC-12 (Lane3), PC-12 differentiated to neurons (Lane 4) and tissue extract of Mouse Brain (Lane 5) and Mouse Liver (Lane 6) were electrophoresed using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001).The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Dihydropyridine Receptor alpha-2 in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Dihydropyridine Receptor alpha-2 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Dihydropyridine Receptor alpha-2 in PC12 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Dihydropyridine Receptor alpha-2 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Dihydropyridine Receptor alpha-2 in U251 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Dihydropyridine Receptor alpha-2 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human skeletal muscle tissue. 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 1:100 with a Mouse Monoclonal Antibody recognizing Dihydropyridine Receptor alpha-2 (Product # MA3-921) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized mouse skeletal muscle tissue. 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 Dihydropyridine Receptor alpha-2 (Product # MA3-921) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Dihydropyridine Receptor alpha-2 in C6 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Dihydropyridine Receptor alpha-2 in Neuro-2a cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of Dihydropyridine Receptor alpha-2 in SH-SY5Y cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Dihydropyridine Receptor alpha-2 monoclonal antibody (Product # MA3-921) at a dilution of 1:100 for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Expression of NCX1 and other Ca handling proteins in atrial-specific NCX1 knockout mice. A . Immunoblot (top) of mouse atrial (A) and ventricular (V) homogenate using an antibody to NCX1 in wildtype control (C) and atrial-specific NCX1 KO mice. There is complete absence of NCX1 protein in the immunoblot. The new band in the KO lanes at ~110 kDa represents nonfunctional NCX in KO atria after excision of exon 11 by Cre recombinase [13] . Ventricular expression of NCX1 is unaffected in atrial-specific KO mice. The bottom panel shows immunofluorescence of isolated SAN node myocytes from control (C) and KO hearts. Myocytes were co-immunolabled with antibodies against HCN4 and NCX1. Both control and KO SAN cells stained positive for HCN4, but only control SAN cells showed staining of NCX at the membrane. B . Immunoblots of sarcoendoplasmic reticulum Ca ATPase (SERCA), plasma membrane Ca pump (PMCA; the lower band at 72 kDa represents an active proteolytic fragment), and dihydropyridine receptor (DHPR) in control (C) and NCX1 KO atria. Note the reduction in SERCA and the increase in PMCA and DHPR in KO.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 8 The p.Leu125Pro mutation impairs complex formation of beta 4b with P/Q-type Ca V 2.1 and L-type Ca V 1.2 channels. HEK293T cells were co-transfected with beta 4b -V5 or beta 4b -L125P-V5 expression construct, mouse alpha2delta-1 expression construct and GFP-Ca v 1.2 or GFP-Ca v 2.1 (alpha 1 subunits) expression construct as indicated. beta 4b -V5 or beta 4b -L125P-V5 was immunoprecipitated with V5-coupled Protein G dynabeads. As control, lysates from cells only transfected with beta 4b -V5 or beta 4b -L125P-V5 construct were incubated with an anti-normal mouse IgG antibody coupled to Dynabeads. Co-precipitated GFP-tagged alpha 1 subunit was detected by anti-GFP antibody. beta 4b -V5 proteins were detected by anti-V5-HRP or anti-beta 4 antibody. Ectopically expressed (mouse) and endogenous (human) alpha2delta-1 were detected by anti-alpha2delta-1 antibody (input, right panel). The band at ~55 kDa corresponds to the heavy chain of the IgG antibody and is marked by an asterisk in the immunoblots of the co-immunoprecipitation (IP V5; left panel). A representative blot of four (Ca V 1.2) and three (Ca V 2.1) independent experiments is shown.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 CACNA2D1 and miR-107 are inversely correlated in tissues. (a) This is an immunofluorescence photograph of a LSCC patient (T4N2M0, moderately differentiated, x400). The arrow shows the typical alpha2delta1 expression (red). (b) The images of alpha2delta1 protein expression detected by WB in adjacent normal tissues and LSCC tissues are as follows: A1 represents the adjacent tissue from patient No.1; C1 represents the LSCC tissue from patient No.1; A2 represents the adjacent tissue from patient No.2; and C2 represents the LSCC tissue from patient No. 2, and so on. * P < 0.05. (c and d) qPCR detection of relative expressions of CACNA2D1 and miR-107 in adjacent normal tissues and LSCC tissues (* P < 0.05). It was easily observed that they are inversely related to normal and LSCC tissues. LSCC, laryngeal squamous cell carcinoma; miR-107, microRNA-107; WB, Western blot.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Differentiation properties of alpha 2 delta 1 + TU212 and TU686 cells. Flow cytometry shows the percentage of alpha 2 delta 1 + cells in TU212 and TU686 cells, FACS-purified alpha 2 delta 1 + cells and purified alpha 2 delta 1 + cells cultured in 10% serum-containing medium for 1 week (cultured).