PA3-821A

antibody from Invitrogen Antibodies

Targeting: PPARG NR1C3, PPARG1, PPARG2, PPARgamma

Provider product page for PA3-821A

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [26]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [6]
Flow cytometry [1]
Other assay [15]

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Product number: PA3-821A - Provider product page
Provider: Invitrogen Antibodies
Product name: PPAR gamma Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA3-821A detects peroxisome proliferator activated receptor (PPAR) gamma from human and mouse samples. This antibody is a reproduction of PA3-821, using the same immunizing peptide and new host rabbits. PA3-821A has been successfully used in IF and Western blot procedures. Previous batches of this antibody have been used in gel shift, immunocytochemistry, and immunofluoresence procedures. By Western blot, this antibody detects a 57 kDa protein representing PPAR gamma 2 from differentiated mouse 3T3-L1 cell lysate. Immunolocalization of previous batches gives predominant nuclear staining. The PA3-821A immunizing peptide corresponds to amino acid residues 284-298 from mouse PPAR gamma 2. This sequence is completely conserved in PPAR gamma 1, but exhibits no significant homology with PPAR alpha or NUC1. This sequence is completely conserved in mouse, human, and rat.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

PPARG protein structure - PA3-821A shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (20 µg lysate) of NIH/3T3 (Lane 1), HeLa (Lane 2), K562 (Lane 3), PC-12 (lane 4) and U-87 MG (lane 5). The blots were probed with Anti-PPAR gamma Rabbit Polyclonal Antibody (Product # PA3-821A, 1:500-1:1500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 54 kDa band corresponding to PPAR gamma was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of PPAR gamma-2 was performed by loading 20 µg of indicated whole cell lysate and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% BSA in TBST for 1 hour at room temperature. PPAR gamma was detected at ~57 kDa using a PPAR Gamma rabbit polyclonal antibody (Product # PA3-821A) at a dilution of 1:500 in blocking buffer overnight at 4C on a rocking platform, followed by a Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036) at a dilution of 1:1000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of PPAR gamma-2 was performed by loading 20 µg of indicated whole cell lysate and 5 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-Glycine polyacrylamide gel (Product # WT4202BX10). Proteins were transferred to a nitrocellulose membrane using the G2 Blotter (Product # 62288), and blocked with 5% BSA in TBST for 1 hour at room temperature. PPAR gamma was detected at ~57 kDa using a PPAR Gamma rabbit polyclonal antibody (Product # PA3-821A) at a dilution of 1:500 in blocking buffer overnight at 4C on a rocking platform, followed by a Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036) at a dilution of 1:1000 for at least 30 minutes at room temperature. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34078).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PPAR gamma (green) showing positive staining in the nucleus and cytoplasm of 3T3-L1 cells (right) compared with a negative control in the absence of primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes, blocked with 3% BSA-PBS for 30 minutes at room temperature and probed with a PPAR gamma polyclonal antibody (Product # PA3-821A) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight 488-conjugated goat-anti-rabbit IgG secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with DAPI for 5-10 minutes in the dark. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PPAR gamma (green) showing positive staining in the nucleus and cytoplasm of Hela cells (right) compared with a negative control in the absence of primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes, blocked with 3% BSA-PBS for 30 minutes at room temperature and probed with a PPAR gamma polyclonal antibody (Product # PA3-821A) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight 488-conjugated goat-anti-rabbit IgG secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with DAPI for 5-10 minutes in the dark. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PPAR gamma (green) showing positive staining in the nucleus and cytoplasm of NIH-3T3 cells (right) compared with a negative control in the absence of primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes, blocked with 3% BSA-PBS for 30 minutes at room temperature and probed with a PPAR gamma polyclonal antibody (Product # PA3-821A) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight 488-conjugated goat-anti-rabbit IgG secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with DAPI for 5-10 minutes in the dark. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of PPAR Gamma was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with PPAR gamma Rabbit Polyclonal Antibody (Product # PA3-821A) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of PPAR gamma-2 (red) in 3T3-L1 differentiated day 7 cells. The cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a PPAR gamma-2 rabbit polyclonal antibody (Product # PA3-821A) at a dilution of 1:200 in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Rabbit IgG (H+L) Secondary Antibody, Dylight 680 (Product # 35568) at a dilution of 1:1000 for at least 30 minutes at room temperature in the dark (red). Lipids (green) were stained with HCS LipidTOX neutral lipid stain (Product # H34475) at a dilution of 1:200 for at least 30 minutes at room temperature in the dark. Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a EVOS FL Auto Imaging System at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PPAR gamma-2 (green) in 3T3-L1 Undifferentiated (left panel) and Differentiated day 7 cells (right panel. The cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a PPAR gamma rabbit polyclonal antibody (Product # PA3-821A) at a dilution of 1:200 in blocking buffer for 1 hour at room temperature, and then incubated with a Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:1000 for at least 30 minutes at room temperature in the dark. Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a EVOS FL Auto Imaging System at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG. 4. Expression of FABP4 and PPAR-gamma proteins in 3D ColI composites after 28 days of culture. Immunofluorescence was visualized by MPLSM. FABP4 (a-c) and PPAR-gamma (d-f) . Adipogenic protein markers (green) and nuclei (blue); scale bar 50 mum.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 ( A ) PPAR-gamma fluorescence (green) with DAPI nuclear counterstain (blue) and quantification ( B ) show a decrease in intensity with increasing stiffness. ( C ) mRNA expression is also inversely related with substrate stiffness. The highest expression and fluorescence and mRNA levels appears on the softest substrates. * indicates p < 0.05 when comparing sham vs. ACF. + indicates a significant effect of substrate stiffness.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 DPP increased transcriptional activity of PPARalpha and PPARgamma. For luciferase, the 3x PPRE-TK-LUC plasmid and PPARalpha ( A ) or PPARgamma ( B ) expression vector were transfected into Ac2F cells. Twenty-four hours after the transfection, the cells were treated with DPP or agonists (WY14643 10 uM and Rosiglitazone 10 uM, respectively) for 5 h. Values are expressed as the mean +- SEM of two independent replications. ( C ) The nuclear levels of PPARalpha and PPARgamma in Ac2F cells were analysed by western blot and band intensity were quantified by ImageJ program. Transcription factor II B (TFIIB) was used as loading control. Statistical results of one-factor ANOVA followed by the Bonferroni test: ** p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Nuclear binding assays. A , a partial SetD7 mouse promoter sequence curated using NM_080,793 as DNA reference sequence. The SetD7 PPRE sequence is bolded and underlined . The DNA sequences used for the primers designed for the ChIP assay PCR are bolded and italicized . B , consensus PPRE sequence on the SetD7 promoter: A putative PPARgamma response element was found using MatInspector software on the SetD7 promoter region that was conserved between mouse, rat, and human SetD7 promoter sequences. C , EMSA and super-shift assay of SetD7 PPRE: PAGE purified infrared dye (IRD-700) tagged oligos for SetD7 PPRE were annealed and EMSA was performed using nuclear extracts prepared from INS-1 cells as detailed in the Experimental procedures section. For the super-shift assay, INS-1 cell nuclear extracts were preincubated with a PPARgamma specific antibody or mouse nonimmune serum on ice for 30 min before adding infrared dye (IRD-700) tagged oligos for SetD7 PPRE. D , upper panel , ChIP assay PCR showing 144 base pairs (bp) SetD7 PPRE positive band: 300-400 bp chromatin preparations of betaTC6 cells were prepared as described in the Experimental procedures section. The prepared chromatin samples were immunoprecipitated using anti-PPARgamma versus a control nonimmune IgG, followed by PCR of the immunoprecipitated and nonimmunoprecipitated DNA (input DNA) using flanking primer pairs to the mouse SetD7-PPRE. The shown representative bands are the 144 bp (expected length) PCR prod

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 SetD7 is reduced in PPARgamma KD and PANC PPARgamma KO beta-cells. A , i , 10x overview of live INS-1 cells expressing GFP-tagged PPARgamma shRNA or scrambled (Scr) shRNA. A , ii , representative WB and band quantitation for PPARgamma, SetD7 and Pdx1 relative to the scrambled control, * p < 0.001. B , i , Representative immunofluorescence fields, imaged were under identical conditions, from 8-week-old WT and PANC PPARgamma KO islets stained for insulin ( green ), SetD7 ( magenta ), and glucagon ( red ). B , ii , representative WB showing protein levels of PPARgamma and SetD7 in the islets of 8-week-old WT and PANC PPARgamma KO mice. B , iii , quantitative band analysis of SetD7 in WT and PANC PPARgamma KO mice. * p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 SetD7 expression in beta-cells of sham and 60% Px in rats post 14-days surgery. A , SetD7 immunohistochemistry: Pancreatic sections of sham and 60% Px rats were stained for SetD7 (indirect immunoperoxidase staining). Representative pancreatic islet fields demonstrate enhanced SetD7 staining in the 60% Px islets compared with sham controls correlating with beta-cell functional compensation. B , SetD7 immunofluorescence analysis: Representative pancreatic islet fields from sham control and 60% Px rats were immunolabeled for insulin ( green ), glucagon ( red ), SetD7 ( magenta ), and nuclei (DAPI, blue ) demonstrating enhanced nuclear SetD7 staining in beta-cells of 60% Px rats compared with sham controls under conditions of sustained beta-cell compensation. C , WB and band quantitation: Lysates from isolated islets from the pancreas of sham & 60% Px rats were analyzed for PPARgamma and SetD7. The data are representative of three separate WBs and demonstrate enhanced expression of PPARgamma and SetD7 in islets of Px rats compared with sham controls. * p < 0.002.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. PPARg protein expression in human spermatozoa based on flow cytometric analysis. Percentage of PPARg protein-positive spermatozoa in normozoospermic (n = 39) and asthenozoospermic (n = 40) groups after flow cytometric analysis (mean+-SEM). The proportion of sperm positive for PPARg protein tended to be higher in normozoospermic than in asthenozoospermic men (P = 0.066).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8. Rosiglitazone effect on nuclear translocation of PPARgamma and CAT transcription in JEG-3 cells. (A) Immunofluorescence proximity ligation assay (PLA) for global transcriptional activity of PPARgamma in JEG-3 cells. Using primary antibodies against PPARgamma and RNA polymerase-2 (POL2), the technique enables visualization of molecular proximity (potentially at gene promoters) at single molecule resolution. Negative control (negative) imaged cells processed after treatment with rosiglitazone (Rosi.) without primary antibodies. (B) ChIP assays demonstrating the occupancy of PPARgamma and RNA polymerase-2 (POL2) on the promoters of the CAT gene. The DNA fragments were IP using specific antibodies against PPARgamma, POL2, or control IgG, and analyzed by qPCR. Input DNA served as an internal control. The target promoter region upstream of the transcription start site of the human CAT gene (verified by MEME SUITE; upper panel) was targeted. (C) Results were normalized to input DNA, and the enrichments were calculated relative to untreated (N/T) control. (D) RT-qPCR was performed in parallel to measure CAT mRNA expression. N = 3; * P < 0.05 vs N/T control, N/T = no-treatment, Rosi. = Rosiglitazone, scale bar = 100 mum, bars present mean +- SEM.