α pparγ

Manufactured by Santa Cruz Biotechnology

Sourced in United States

α-PPARγ is a laboratory reagent used in research applications. It is an antibody that specifically binds to and detects the PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) protein, which is a nuclear receptor that regulates the expression of genes involved in adipogenesis, glucose metabolism, and inflammation.

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4 protocols using α pparγ

Immunoprecipitation and Western Blotting Analysis

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Immunoprecipitation was done by following the procedure described in earlier studies;^{20 (link)} In brief, equal amounts (750 μg) of cell lysates obtained after the indicated treatments were incubated with α-RUNX2 or α-p-AMPK (cell Signaling) at 4 °C overnight. Protein A-Sepharose resin beads (2.5 mg/sample) (GE Healthcare) were used to pull the immunoprecipitated protein complexes and were subjected to western blotting. Where immunoprecipitation was not done, cell lysates were directly subjected to western blotting and probed with respective specific antibodies α-RUNX2, α-β-Actin, α-p-AMPK, α-Bip1, α-p-GSK3β, α-p-JNK, α-p-AMPK substrate motif-specific antibodies and respective horseradish peroxidase enzyme-linked secondary antibodies obtained from Cell Signaling Technology, USA. The α-PPARγ was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The signal was detected by the Amersham ECL prime Western blotting detection reagent, and images were documented by Bio-Rad chemidoc MP system.

Chava S., Chennakesavulu S., Gayatri B.M, & Reddy A.B. (2018). A novel phosphorylation by AMP-activated kinase regulates RUNX2 from ubiquitination in osteogenesis over adipogenesis. Cell Death & Disease, 9(7), 754.

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PPARγ-TRIM25 Interaction Characterization

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Glutathione S-transferase (GST)-fused proteins (PPARγ domain mutants) immobilized with glutathione-agarose were incubated with TRIM25-expressing cell lysates for 2 h at 4 °C. Protein complexes were pulled down by centrifugation and washed four times with binding buffer. Precipitates were detected by immunoblotting using anti-GST or TRIM25 antibodies. For analyzing interactions between endogenous PPARγ and TRIM25, 3T3-L1 adipocytes were lysed with binding buffer. Cell lysates were incubated with anti-PPARγ or TRIM25 antibodies and analyzed by western blotting. HEK-293 cells expressing PPARγ, TRIM25, or their mutants were lysed in binding buffer, and total cell lysates were incubated with an anti-hemagglutinin (HA) antibody at 4 °C. Immunoprecipitants or total cell lysates were analyzed with specific antibodies as indicated. The antibodies used in this study included α-TRIM25, α-PPARγ, α-GST, α-Ub, α-aP2, and α-adipsin antibodies, which were purchased from Santa Cruz Biotechnology (Dallas, TX), while α-HA, α-actin, α-HSP90, and α-adiponectin were purchased from Cell Signaling Technology (Danvers, MA).

Lee J.M., Choi S.S., Lee Y.H., Khim K.W., Yoon S., Kim B.G., Nam D., Suh P.G., Myung K, & Choi J.H. (2018). The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPARγ. Experimental & Molecular Medicine, 50(10), 135.

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Immunoprecipitation and Western Blot Analysis

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Cell lysates were incubated overnight at 4 °C with the indicated antibodies diluted 1:200, and added to protein A/G agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA). Immune complexes were released from the beads by boiling, and then analyzed by WB. Primary antibodies used in this study: α-FLAG (F1804; Sigma-Aldrich, St. Louis, MO, USA), α-Myc (05-724MG; Millipore, Billerica, MA, USA), α-GFP (sc-9996; Santa Cruz Biotechnology), α-SIRT1 (sc-15404; Santa Cruz Biotechnology), α-PPARγ (sc-7273; Santa Cruz Biotechnology), α-β actin (sc-47778; Santa Cruz Biotechnology), α-LSD1 (ab17721; Abcam, Cambridge, UK), α-H3K9ac (ab12179; Abcam), α-H3K9me2 (ab1220; Abcam), and α-CACUL1 (rabbit polyclonal antibody raised against amino acids 338–355; Peptron, Daejeon, South Korea). The WEST-ZOL^® system (iNtRON Biotechnology, Seongnam-Si, South Korea) was used for detecting protein bands.

Jang M.J., Park U.H., Kim J.W., Choi H., Um S.J, & Kim E.J. (2017). CACUL1 reciprocally regulates SIRT1 and LSD1 to repress PPARγ and inhibit adipogenesis. Cell Death & Disease, 8(12), 3201.

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Comprehensive Antibody Evaluation for Cell Signaling

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Antibodies against α-Rab5a (#2143), α-Rab7a (#9367), α-Rab10 (#8127), α-EGFR (#4267), α-Phospho-EGF Receptor (Tyr1068) (#2234 S), α-Phospho-p38 MAPK (Thr180/Tyr182) (#2775 S), α-p38 MAPK (D13E1) XP Rabbit mAb (#8690), α-p44/42 MAPK (Erk1/2) (#695), α-IRG1 (#17805) were purchased from Cell Signaling Technology. Antibodies against α-HIF1-α (sc-53546), α-EGFR (sc-120), α-PKM2 (sc-365684), α-LDHA (sc-133123), α-MAPK14 (sc-11415), α-EGFR (sc-120), α-EEA1 (sc-137130), α-TGN38 (sc-166594), α-LAMP1 (sc-20011), α-PPARγ (sc-7273), α-SDHA (sc-390381), α-COX2 (sc-514489), α-COX4 (sc-517553), α-ATP5A (sc-136178) were purchased from Santa Cruz Biotechnology. Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group. Antibodies against APC anti-mouse iNOS (#85-17-5920-82, eBioscience), PE anti-mouse CD206 (MMR) Antibody (#85-12-2061-82, eBioscience), α-F4/80 (11-4801-82, FITC, eBioscience), PE anti-mouse Ly6G (#12-9668-82, eBioscience), α-CD14 (25-0149-42, FITC, eBioscience) were purchased from eBioscience. Antibodies against α-GAPDH (T0004), α-β-Tubulin (T0023), α-Phospho-PPAR gamma (Ser112) (AF3284), α-Phospho-PKM2 (AF7231) were purchased from Affinity Biosciences.

Zhang X., Chen C., Ling C., Luo S., Xiong Z., Liu X., Liao C., Xie P., Liu Y., Zhang L., Chen Z., Liu Z, & Tang J. (2022). EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis. Cell Death & Disease, 13(11), 934.

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