Anti hif1a

Samples were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors and were diluted to a concentration of 20 μg of protein and heated at 99°C for 5 min with a denaturation buffer. Proteins were separated by SDS–PAGE electrophoresis and transferred to PVDF membranes (Amersham International). Membranes were incubated in blocking reagent (PBS with 0.5% Tween 20 and 3% of bovine serum albumin) for 30 min, then in primary antibody (in the blocking solution) overnight at 4°C. The antibodies and their concentrations are the following: anti-HIF1a (Novus, NB100-105; 1:500), anti-β-actin (Abcam, ab8226; 1:30000), anti-phospho-AKT (Cell signaling, 9331S; 1:1000), anti-AKT (Cell signaling, 9272S; 1:1000), anti-Flag (Sigma-Aldrich, F3165; 1:5000), anti-HSL (cell signaling, 4107; 1:1000), anti-phospho-HSL (ser660) (cell signaling, 4126; 1:1000), anti-ATGL (cell signaling, 2138; 1:1000) and custom-made anti-GPS2 N/C ((Fan et al., 2016 (link)). After several washes in PBS with 0.5% Tween 20, membranes were incubated in horseradish peroxidase (HRP)-labeled secondary antibodies (1:10000) for 1 h at room temperature in the blocking solution. Membranes were incubated with ECL western-blotting substrate (Pierce, Ref# 32106) and imaged by myECL Imager (ThermoFisher).