PA1-650

antibody from Invitrogen Antibodies

Targeting: ABCD3 PMP70, PXMP1, ZWS2

Provider product page for PA1-650

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [29]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [7]
Flow cytometry [3]
Other assay [11]

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Product number: PA1-650 - Provider product page
Provider: Invitrogen Antibodies
Product name: PMP70 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA1-650 detects peroxisomal membrane protein 70 (PMP70) from mouse tissues. PA1-650 has been successfully used in Western blot, flow cytometry, and immunofluorescence procedures. By Western blot, this antibody detects an ~70 kDa protein representing PMP70 from peroxisome-enriched fractions from L cells. PA1-650 immunizing peptide corresponds to amino acid residues 644-659 from rat PMP70. This sequence is completely conserved in mouse PMP70 and differs from human PMP70 by a single amino acid substitution. PA1-650 immunizing peptide (Cat. # PEP-038) is available for use in neutralization and control experiments.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

ABCD3 protein structure - PA1-650 shown in red.

Supportive validation

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Experimental details: Immunofluorescent analysis of PMP70 (green) in 3T3 cells. The cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes, and blocked with 3% Blocker BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a PMP70 rabbit polyclonal antibody (Product # PA1-650), at a dilution of 10 µg/mL in blocking buffer for at least 1 hour at room temperature, and then incubated with a Goat anti-Rabbit IgG Superclonal secondary antibody, Alexa Fluor 488 conjugate (Product # A27034) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PMP70 using anti-PMP70 polyclonal antibody (Product # PA1-650) shows staining in A549 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PMP70 using anti-PMP70 polyclonal antibody (Product # PA1-650) shows staining in HMVEC Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PMP70 using anti-PMP70 polyclonal antibody (Product # PA1-650) shows staining in NS-1 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PMP70 using anti-PMP70 polyclonal antibody (Product # PA1-650) shows staining in p19 Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of PMP70 was performed using 70% confluent log phase A-431 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 1 hour at room temperature. The cells were labeled with PMP70 Rabbit Polyclonal Antibody (Product # PA1-650) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, Alexa Fluor 488 (Product # A27034) at a dilution of 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). Panel d represents the merged image showing cytoplasmic (peroxisomal pattern) localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Knockdown of PMP70 was achieved by transfecting A-431 cells with PMP70 specific siRNA (Silencer® select Product # s11616). Immunofluorescence analysis was performed on A-431 cells (untransfected, panel a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with PMP70 specific siRNA (panel c,f). Cells were fixed, permeabilized, and labelled with PMP70 Rabbit Polyclonal Antibody (Product # PA1-650, 5 µg/mL), followed by Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 (Product # A27034, 1:2000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Reduction of specific signal was observed upon siRNA mediated knockdown (panel c,f) confirming specificity of the antibody to PMP70 (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of PMP70 in 293T cells (green) 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 PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 0.5 µg/test 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-rabbit 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 PMP70 in HepG2 cells (green) 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 PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 0.5 µg/test 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-rabbit 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 PMP70 in NIH-3T3 cells (green) 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 PMP70 polyclonal antibody (Product # PA1-650) at a dilution of 0.5 µg/test 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-rabbit IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 2 Autophagy inhibitors do not restore peroxisomal import of PTS1- and PTS2-targeted peroxisomal matrix proteins in primary PEX1-G843D fibroblasts. Immunoblot analysis of homogenates of primary fibroblasts from two PBD-ZSD patients homozygous for the PEX1-G843D mutation [PEX1-G843D(1) (A) and PEX1-G843D(2) (B) ] treated as described in Figure 1A . The protein levels of the PMPs ACBD5 and ABCD3 and the ratio of the processed over unprocessed forms of PTS1-targeted ACOX1 and PTS2- targeted thiolase were determined using specific antibodies. Below the ACOX1 bands, the corresponding ratios between the intensity of processed (50 kDa) over unprocessed (70 kDa) ACOX1 bands are indicated. Below the thiolase bands, the corresponding ratios between the intensity of processed (41 kDa) over unprocessed (44 kDa) thiolase bands are indicated. Below the ACBD5 and ABCD3 bands, the corresponding signal intensity relative to the tubulin band intensity, which was used as a loading control, is indicated. Homogenates of non-treated control fibroblasts and PEX1- fibroblasts (PEX1 -/- ) were used for reference. The effect of the different treatments on autophagy was assessed by determining the relative protein levels of the autophagy adaptor protein SQSTM1/P62 (P62; relative to tubulin levels) and the ratio of MAPILC3B-II (LC3-II) over MAPILC3B-I (LC3-I). Cells were incubated for 7 days with the different compounds, except for bafilomycin (24 h incubation). Experiments were repeated twice

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 Autophagy inhibitors do not restore import of peroxisomal matrix proteins and metabolic functions in transformed-and-immortalized patient cells. The effect of treatment with different compounds on peroxisomal matrix protein import and peroxisomal metabolic functions was assessed in previously reported PEX1-G843D fibroblasts that had been transformed and immortalized (tr/immPEX1-G843D) () as well as in the same cells stably overexpressing GFP-PTS1 (tr/immPEX1-G843D + GFP-PTS1) (). Cells were treated with 20 mM L-arginine (L-arg), 5% glycerol (Gly), 10 muM hydroxychloroquine (HCQ), or 10 mM 3-methyladenine (3-MA) for 24 h. (A) Quantification of tr/immPEX1-G843D cells that display functional peroxisomal catalase import after treatment. Cells were analyzed by immunofluorescence microscopy to determine the subcellular localization of catalase (> 150 cells analyzed for each condition). (B) Density of ABCD3 punctated structures (indicating peroxisomal membranes) per cell area determined in treated tr/immPEX1-G843D cells using immunofluorescence microscopy (see Materials and Methods for more details). Data is presented as mean of n > 45 cells +- SD. (C) Immunoblot analysis of homogenates of tr/immPEX1-G843D cells, which were treated for 7 days with the different compounds. The protein levels and processing of thiolase and the PMPs ACBD5 and ABCD3 were determined using specific antibodies. Below the thiolase bands, the corresponding ratios between the intensity of processed (

Supportive validation

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Experimental details: Fig 4 KSHV latently infected endothelial cells induces peroxisome formation. (A.) Flow cytometry of mock- and KSHV- infected TIME cells (TIMECs) harvested at 48 hpi, fixed and stained with antibody to ABCD3, a peroxisome marker. (B.) Geometric mean fold change of KSHV over mock at 48hpi for 3 experiments as in panel A, p < 0.05 student's t-test. (C.) Flow cytometry of mock- and KSHV- infected TIMECs, harvested at 96 hpi, fixed and stained as in panel A. (D.) Geometric mean Fold change of KSHV over mock at 96 hpi for 3 experiments as in panel C, p < .05 student's t-test. (E.) Flow cytometry of mock- and KSHV- infected primary human dermal microvascular endothelial cells (hDMVECs) harvested at 96 hpi, fixed and stained as in A. (F.) Geometric mean fold change of KSHV over mock at 96 hpi for 3 experiments as in E, p < .05 student's t-test. (G.) Flow cytometry of mock- and KSHV- infected lymphatic endothelial cells (LECs) harvested at 96 hpi, fixed and stained as in A. (H.) Geometric mean fold change of KSHV over mock at 96 hpi for 3 experiments as in panel B, p < .05 student's t-test. All the data are represented as mean +/- SEM and were analyzed using FlowJo software. (I) Representative confocal images of Mock and KSHV infected TIME cells at 96 hpi stained with antibody to ABCD3 and DAPI to identify the nuclei. (J) Quantification of number of peroxisomes per cell in three biological replicates of Mock and KSHV-infected cells stained as in panel I, analyzed using student's t-

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 10.1371/journal.ppat.1006256.g005 Fig 5 KSHV latency locus is sufficient to induce a peroxisome marker in endothelial cells. (A.) Flow cytometry of mock-, KSHV-UV irradiated and KSHV- infected TIME cells harvested at 96 hpi, fixed and stained with antibody to ABCD3. (B.) Geometric mean fold change of KSHV, KSHV-UV-irradiated over mock at 96 hpi for 3 experiments as in A, p < .05 student's t -test. (C.) Flow cytometry of AdGFP or AdKLAR (KSHV latency-associated region in a gutted adenovirus) infected TIME cells harvested at 96 hpi, fixed and stained with antibody to ABCD3. (D.) Geometric mean fold change of AdGFP over AdKLAR at 96 hpi p < .05 student's t-test. All the data are represented as mean +/- SEM and were analyzed using FlowJo software. (E.) Western blot analysis of TIME cells mock infected or infected with AdGFP-, or AdKLAR stained with antibodies to GFP or LANA.

Supportive validation

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Experimental details: 10.1371/journal.pbio.3000988.g005 Fig 5 Cool adaptation of adipocytes stimulates beta-oxidation of fatty acids and increases number and functioning of mitochondria. ( A, B ) Adipocytes at indicated days of cool temperature exposure ( A ; n = 5) or for 12 days ( B ; n = 6) were incubated with tritiated palmitate ( A ) or oleate ( B ) for 3 hours +/- 40 muM etomoxir. * significantly different from day 0 control at 37degC; p < 0.05). t indicates a difference from day 0 etomoxir treatment. ( C ) Cool adaptation increases mitochondrial fatty acid oxidation and peroxisomal proteins. ( D ) Primary adipocytes isolated from eWAT or sWAT were cultured in suspension at either 37degC or 31degC for 2 days. Expression of SCD1, CPT1alpha, MCKAT, and MTPbeta was then determined by immunoblot analyses; adiponectin, FABP4, and laminin were loading controls. ( E ) Elevated basal OCR of adipocytes at 31degC is partially dependent on oxidation of endogenous fatty acids. MSC-derived adipocytes cultured at 31degC or 37degC for 12 days were treated with vehicle, 20 muM etomoxir and/or 20 muM of atglistatin for 2 hours prior and during assay of OCR ( n = 8-10). ( F ) Four-day exposure at 31degC stimulates an increase in mitochondrial number as assessed by qPCR for 2 mitochondrial regions (CytoB, Cox-TMS) and 2 nuclear genes (glucagon, beta-globin; n = 3). ( G - I ) Isolated mitochondria from adipocytes adapted to 31degC have improved functioning of complex I and II. Mitochondria were incubated with m

Supportive validation

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Experimental details: Analysis of peroxisomes distribution in CFBE41o- cells, parental and expressing both F508del and WT-CFTR, under control condition and following treatment with VX-809. ( A ) Microphotographs showing peroxisomes distribution as evidenced by staining for the peroxisomal markers PMP70 and PEX13 in cells treated either with vehicle alone (DMSO) or with VX-809 (3 uM). PMP70 was stained using a rabbit anti-PMP70 as primary antibody followed by an anti-rabbit secondary antibody conjugated to AlexaFluor 488. PEX13 was stained using a mouse anti-PEX13 as primary antibody followed by an anti-mouse secondary antibody conjugated to AlexaFluor 647. Cell nuclei were counterstained with Hoechst 33342. ( B ) Quantification of the number of PMP70- (left graph) and PEX13- positive (right graph) peroxisomes per cell, in cells treated with DMSO vs. VX-809. Each dot represents the value obtained from the analysis of a different biological replicate, in which 700cells were imaged and analyzed. Lines indicate means +- SD, n = 16. Statistical significance of VX-809 treatment was tested using a Student's t -test. Symbols indicate statistical significance versus DMSO: *** p < 0.001. Scale bar = 25 um. Only statistically significant comparisons are reported.

Supportive validation

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Experimental details: Analysis of mitochondrial network morphology and peroxisomal distribution in CF and non-CF bronchial epithelia. Well-differentiated bronchial epithelia were generated from two F508del/F508del homozygous CF patients (donor ID: BE49 and BE55) and two non-CF subjects (donor ID: BE165 and BE168). ( A ) Microphotographs showing mitochondrial network as evidenced by staining for the mitochondrial marker TOMM20 in epithelia treated for 24 h either with vehicle alone (DMSO) or with VX-809 (3 uM). ( B ) Scatter dot plots showing results of TOMM20 signal texture analysis (based on SER features). Each dot represents the value obtained from the analysis of one region of 1050 um x 700 um. Lines indicate means +- SD, n = 6. ( C ) Microphotographs showing peroxisomes distribution as evidenced by staining for the peroxisomal markers PMP70 and PEX13 in epithelia treated for 24 h either with vehicle alone (DMSO) or with VX-809 (3 uM). ( D ) Quantification of the calculated percentage of PEX13-positive peroxisomes in each image field. Each dot represents the value obtained from the analysis of one region of 1050 um x 350 um. Lines indicate means +- SD, n = 9. Statistical significance of VX-809 treatment was tested using a Student's t -test. Symbols indicate statistical significance versus DMSO: *** p < 0.001; ** p < 0.01. Scale bar = 40 um. Only statistically significant comparisons are reported.

Supportive validation

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Experimental details: 6 FIG. Altered hepatic nuclear receptor expression and FXR agonists in malnutrition. (A-C) Relative concentrations of FXR and PPARalpha protein in liver, determined by western blot, reveal a profound decrease of PPARalpha expression in malnourished male livers (n = 3-4). (D) Relative expression of PMP70 correlates directly with PPARalpha protein levels in malnourished male livers (n = 3-4) and supports qualitative evaluation by electron microscopy revealing loss of hepatocyte peroxisomes (Supporting Fig. S8). (E) Targeted bile acid mass spectrometry from liver, plasma, and stool found total fecal bile acid levels to be significantly decreased in malnourished male mice. (F) Individual stool bile acid metabolites show multiple decreased bile acids and sex specificity (n = 6). Individual liver and plasma bile acid metabolites are shown in Supporting Fig. S9. Data in (B-D) are mean + SD. Box and whisker plots (E,F) show interquartile range (box), median (vertical line), and outliers (whiskers). Student t test; **** P < 0.0001, ** P < 0.01, * P < 0.05 compared to control mice of the same sex. [Corrections added on October 24, 2020 after first online publication: the figure 6 has been replaced with the correct one.]