Anti pparγ antibody

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and newborn calf serum (BCS) were purchased from Gibco Life Technologies (Grand Island, NY, USA), Insulin, indomethacin, dexamethasone, 3-isobutyl-1methylxanthine, and Oil Red O solution were purchased from Sigma-Aldrich (St Louis, MO, USA). 3-(4,5)-dimethylthiazo-2-yl-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). Primers were purchased from Macrogen (Seoul, Korea). Anti-C/EBPα antibody and anti-β-actin antibody were purchased from Cell Signaling Technology (Beverly, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Anti-PPARγ antibody and anti-adiponectin antibody were purchased from Abcam (Cambridge, UK).

ChIP assays were carried out with an EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. Immunoprecipitations were carried out using anti-PPARγ antibody (Abcam, A3409A) and an isotype-matched IgG as the control. Reporter assays were performed in 12-well plates using PPRE-Luc (0.5 μg) and pRL-TK (0.2 μg) reporter plasmids, and the luciferase activities were determined using Dual Luciferase Reporter Assay System (Promega).

Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit (Beyotime) according to the manufacturer's instructions. Briefly, 10⁷ neurons were cross‐linked with 1% formaldehyde at 37°C for 10 minutes, and the reaction was stopped with 0.125 mol/L glycine. Cross‐linked neurons were lysed with SDS lysis buffer, followed by sonication to yield DNA fragments of 100‐500 base pairs. After centrifugation, 200 μL of the supernatant was diluted with 1.8 mL of ChIP dilution buffer containing 1 mmol/L PMSF and cleared with 70 μL of Protein A/G agarose/salmon sperm at 4°C for 30 minutes. The cleared supernatant was then incubated with 2 μg of the anti‐PPARγ antibody (Abcam) or 2 μg of mouse IgG (Beyotime), overnight at 4°C. The immunocomplexes were precipitated with 60 μL of Protein A/G agarose/salmon sperm, eluted, and cross‐links were reversed by incubating the complexes at 65°C for 4 hours. Then, the precipitated DNA was cleaned with RNase and proteinase, and purified with a DNA Purification Kit (Beyotime). The DNA fraction derived from ChIP with the anti‐PPARγ antibody was amplified by qRT‐PCR as described above, using primers of promoter region of AZGP1 (Table S3). The results of ChIP were analysed using Percent Input Method.³³

Cells were transfected with HA-Mark4 plasmid, pc-PPARγ plasmid, or both. Cell lysates were obtained in RIPA buffer 48 h after transfection. Mark4 protein and PPARγ protein were immunoprecipitated using the anti-HA antibody (ab9110) and anti-PPARγ antibody separately (Abcam, Cambridge, UK). Mark4 and PPARγ were western blotted using anti-PPAR and anti-HA antibody.
Adipocytes were prepared for chromatin immunoprecipitation (ChIP) assay using a ChIP assay kit (Abcam, England) according to the manufacturer’s protocol. Primary antibodies of PPARγ (ab41928, Abcam, Cambridge, UK) or IgG (ab171870, Abcam, Cambridge, UK) were used. DNA-protein crosslinking complexes were collected, and purified DNA was subjected to qPCR with SYBR green fluorescent dye (Invitrogen, Californian, USA).

J774 cell lysates were prepared using IP lysis buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Roche). Protein A/G agarose beads (Santa Cruz Biotechnology) were added to the cell lysates for 1 h at 4 °C. Thereafter, precleared lysates were incubated with anti-MDM2 antibody (Cell Signaling Technology), TRIM25 (Abcam), RBBP6 (Thermo Fisher Scientific), SIAH2 (Thermo Fisher Scientific), or anti-PPARγ antibody (Abcam) overnight at 4 °C. The next day, Protein A/G Agarose was added to the sample, which was then incubated for 2 h at 4 °C. The immunoprecipitated complexes were eluted by boiling in 2 × Laemmli buffer. The eluted proteins were analyzed through Western blotting to detect MDM2, TRIM25, RBBP6, SIAH2, and PPARγ.