Anti pparg

Following procedures described in (9 (link)), preadipocyte cells were plated at 15,000 cells/well and treated as indicated in 96-well plates (Costar). Cells were fixed with 3% paraformaldehyde in PBS for 30 min. Then the cells were gently washed three times with PBS and permeabilized with 0.05% saponin (Sigma #47036), blocked with 3% BSA (Sigma #7906), and stained with DAPI (1:10,000), anti-PPARG (1:500, Santa Cruz Biotech #sc-7273), anti-CEBPA (1:500, Santa Cruz Biotech #sc-61 or #sc-7962), anti-CEBPB (1:500, Santa Cruz Biotech #sc-150), anti-pAKT (Ser473, Cell Signaling #D9E), or BODIPY 493/503, (1 μg/ml, Molecular Probes #D-3922). Alexa Fluor-514 (#A31558), -555 (#A21429), -594 (#A11032), and -647 (#A31571) (1:1,000, Invitrogen) were used as secondary antibodies. Images were taken using an ImageXpress Micro XL system (Molecular Devices) and analyzed using Cell Profiler software.

Protein extracts (50 μg) from pooled CTR, 45% and 60% HFD, female and male gastrocnemius muscle samples were loaded in duplicate and resolved on 12% gradient polyacrylamide gels. Blots were incubated with rabbit polyclonal anti-Ppargc1a (Santa Cruz Biotechnology, Dallas, TX, USA, sc-13067, 1:500) and anti-Pparg (Santa Cruz Biotechnology, sc-6285, 1:500). After washing, membranes were incubated with anti-rabbit (GE Healthcare, 1:10,000) or anti-goat (Santa Cruz Biotechnology, 1:5000) secondary antibody conjugated with horseradish peroxidase. Signals were visualized by chemiluminescence using the ECL Prime detection kit and the Image Quant LAS 4000 (GE Healthcare) analysis system. Band quantification was performed using the Image Quant TL (GE Healthcare) software followed by statistical analysis (ANOVA + Tukey, n = 2, p-value < 0.05). Band intensities were normalized against the total amount of proteins stained by Sypro ruby total-protein stain.