Anti pparγ antibody

3T3-L1 adipocyte differentiated with the above-mentioned differentiation medium were pretreated with the treatment of compounds for 24 h followed by TNFα (50 ng/mg) for 3 h. Cells were then washed with PBS and lysed with lysis buffer (Cell Signaling Technology, Danvers, MA) containing EDTA (Thermo, IL) and phosphatase inhibitors (Thermo). Aliquots of 20 μg total protein were separated on 7% SDS-PAGE gels (Life Technologies) and transferred to PVDF membranes (Life Technologies). The membranes were probed with primary antibodies followed by the appropriate HRP-conjugated secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG, 1:3000; Santa Cruz Biotechnology, CA, USA). Blots were then developed. The primary antibodies were: anti-Ser-273 PPARγ (Bioss Antibodies) or anti-PPARγ antibody (Santa Cruz Biotechnology).

ChIP was conducted as per our recent publications (Gavin et al., 2012 (link); Gavin et al., 2015 (link)). Cross-linking was performed by adding a final concentration of 1.5% methanol-free formaldehyde to cell culture wells for 5 minutes. Cross-linking was quenched using a final concentration of 130mM of glycine. Cells were washed with PBS in the presence of protease inhibitors, then sonicated to 200–500 bp segments in SDS lysis buffer. Sonicated chromatin was resuspended at 1:10 in ChIP dilution buffer. IP samples were pre-cleared with 20 µL Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Dallas, TX) for 2 hours on a rotator at 4°C. Subsequently, samples were incubated overnight with anti-PPARγ antibody (Santa Cruz, Dallas, TX) on a rotator at 4°C. Antibody-DNA complexes were precipitated using Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Dallas, TX) for 3 hours on a rotator at 4°C. Following low salt, high salt, lithium chloride, and TE washes complexes were eluted from beads at 67°C for 2 hours using elution buffer then IP and input (33% of IP by volume) samples were uncrosslinked using a final concentration of 0.2 M NaCl. DNA was then extracted using proteinase K at 37 °C overnight followed by phenol-chloroform extraction and resuspended in water. Samples were measured using qPCR. IP samples were normalized to input.