Protein Extraction and Western Blot Analysis in Adipocytes

Protein extraction and Western blot analysis were performed as described previously33 (link) with some modifications. Adipocytes and adipose tissues were lysed by dissolution in lysis buffer. The proteins obtained were subjected to sodium dodecyl sulfate–polyacrylamides gel electrophoresis, following which the separated protein bands were transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with the following primary antibodies: anti-PRMT4 (1:2000, Cell Signaling Technology, USA), anti-phosphorylated HSL (1:2000, Cell Signaling Technology, USA), anti-HSL (1:1000, Cell Signaling Technology, USA), and anti-beta (β)-tubulin (1:1000, Cell Signaling Technology, USA). Then, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibodies. The specific bands were detected using enhanced chemiluminescence detection reagents with a Bio-Rad (Hercules, CA, USA) imaging system.