Anti p hsl ser563

Protein lysates were run on 4–15% Gradient Mini-Protean TGX stain-free pre-cast protein gels, followed by transferring the proteins to Nitrocellulose membrane. Unless otherwise stated, a total of 20 μg of protein lysate was loaded per well and nitrocellulose membranes were blocked using 5% (w/v) Non-fat milk in Tris buffered saline with 0.1% (v/v) Tween-20 (TBST) for 4 h at room temperature. Primary antibody incubations were carried out in 5% (w/v) BSA in TBST at the following antibody concentrations: UCP1-Abcam#10983; Anti-pHSL (Ser563) Cell Signaling Technology Cat# 4139; Anti-phospho PKA substrate (RRXS∗/T∗) Cell Signaling Technology Cat# 9624; ERRa-antibody Cell Signaling Cat# 13826, 1:1000; Vinculin Cell Signaling Technology Cat# 18799, OXPHOS Abcam #110413, Tubulin Sigma Cat# T5168. Blots and primary antibodies were incubated overnight with a roller mixer at 4 °C. Membranes were washed with TBST prior to secondary antibody incubations. HRP-conjugated secondary antibodies were diluted with 5% BSA (w/v) in TBST at 1:10,000 for 45 min at room temperature with constant shaking. Membranes were washed in TBST, followed by incubating with Perkin Elmer Western Lightning Enhance ECL. The Bio-Rad Chemi-Doc XRS was used to image the chemiluminescence and quantifications were performed using the system software, or Image J v.1.51.