Molecular imager chemidoc xbs imaging systems

Tissues were homogenized using lysis buffer (keyGEN Bio TECH, China), containing protease and phosphatase inhibitors and centrifuged at 12,000 × g, 4°C for 5 min. The supernatant was collected where protein concentration was measured using the BCA Protein Assay kit (keyGEN Bio TECH, China), according to the manufacturer's instructions. The SDS-PAGE protein loading buffer (5×) (Beyotime, China) was added to the sample and heated at 100°C for 5 min to fully denature the protein. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane and then blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at 4°C with primary antibodies targeting phosphorylated hormone sensitive lipase (p-HSL) (ser563) (1 : 1000, #4139, Cell Signaling Technology, USA), hormone sensitive lipase (HSL) (1 : 1000, ab45422, abcam, USA), adenosine 5'-monophosphate- (AMP-) activated protein kinase (AMPK) (1 : 1000, ab3760, abcam, USA), and phosphorylated AMPK (p-AMPK) (1 : 1000, ab133448, abcam, USA). Then, the blots were incubated with HRP-conjugated secondary antibodies and detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) using Molecular Imager ChemiDOC™ XBS imaging systems (Bio-Rad Laboratories).