Pparγ sc 7196

For Western blot analyses, 8 μg of protein were loaded in each lane. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, El Prat de Llobregat, Spain) overnight at 4°C and 100 mA. Reversible Ponceau staining was used as a loading control (39 (link)). The PVDF membranes were blocked with 5% skimmed milk powder in Tris-Buffered Saline and Tween 20 (TBS-T) for 1 h 15 min and probed with rabbit polyclonal Pparγ (sc-7196) and goat polyclonal Gapdh (sc-20357) primary antibodies (Santa Cruz Biotechnology, CA, USA) at a dilution of 1:200 overnight at 4°C on a tube rotator. Membranes were washed in TBS-T and probed with a horseradish peroxidase-conjugated anti-rabbit (sc-2004) or anti-goat (sc-2020) secondary antibody (Santa Cruz Biotechnology, CA, USA) at a dilution of 1:10,000 for 1 h 15 min. Membranes were washed with TBS-T and chemiluminescent detection performed using an enhanced chemiluminescence kit (Pierce ECL Western blotting Substrate; Thermo Scientific, Alcobendas, Spain). Western blotting results were obtained from two independent cultures, and band intensities were quantified by scanning densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to Ponceau staining.