Prdm16

Cellular proteins were isolated from cells at various time points for each group, and immunoblotting was performed as previously described [17 (link)]. The sources of primary antibodies used in this study are as follows: UCP1 (Abnova, USA), PGC-1α, AMPK, pAMPK, PPARγ (Cell Signalling Technology), AKT (Abcam, USA), phosphorylated AKT (pAKT, Thr308, Bioworld, USA), PRDM16, SIRT1 and C/EBPβ (Santa Cruz Biotechnology, USA). Protein levels were normalized to α-tubulin (GenScrept, USA).

Cells or tissue was lysed in lysis buffer (iNtRON Biotechnology, Seoul, Korea) supplemented with phosphatase and protease inhibitors. The total protein contents of the lysates were quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA) and then equalized. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. These membranes were blocked using 5% non-fat dried milk for 1 h and then incubated overnight at 4 °C with primary antibodies targeting AMPKα, phospho-AMPKα (Thr172), phosphor-hormone-sensitive lipase (p-HSL, Ser563), adipose triglyceride lipase (ATGL, Cell Signaling Technology), fatty acid-binding protein 4 (FABP4), CCAAT enhancer-binding protein α (C/EBPα), diacylglycerol acyltransferase 1 (DGAT1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), PGC1α, peroxisome proliferator-activated receptor alpha (PPARα), PRDM16 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), sterol regulatory element-binding transcription factor 1 (SREBP1), CPT1, or UCP1 (Abcam, Cambridge, UK). After washing, the membranes were incubated for 4 h with secondary antibodies conjugated with horseradish peroxidase (1:1000, Santa Cruz Biotechnology) in 5% non-fat dried milk. Reactive band was obtained by chemiluminescence and LAS image software (Fuji, Valhalla, NY, USA).