Image lab upgrade for chemidoc xrs system 170 8299

For immunoprecipitation, 1 mg of tissue lysate was incubated with 2 μg of antibody (anti-SREBP1c) at 4 °C overnight. After the addition of 40 μl PureProteome™ Protein A/G Mix Magnetic Beads (Millipore), incubation was continued for an additional 2 h at 4 °C. The beads were then collected by magnet and washed three times with washing buffer (PBS containing 0.1% Tween 20). 2x SDS sample buffer was added to the beads and incubated at 95 °C for 10 min, and Western blot was then performed. Acetylated SREBP1c was detected by an anti-acetylated lysine antibody (Cell Signaling, Danvers, MA, USA).
Western blot was performed as described previously14 (link) to detect precursor and mature forms of SREBP1c (Santa Cruz Biotechnologies, Santa Cruz, CA), pAMPK, AMPK, p-p38, p38, SCD1, Sirt1, acetylated-lysine (Cell Signaling Technologies), FGF21 (Abcam, San Francisco, CA), PPARα, PGC1α, β-actin (Santa Cruz Biotechnology), p-PGC1α (R&D, Minneapolis, MN). Blots were scanned using a Bio-Rad Imaging System (Image Lab™ Upgrade for ChemiDoc™ XRS + System #170-8299). All specific bands were quantified with the Automated Digitizing System (Image Lab 4.1). Results are representative of three independent experiments.