Western Blot Analysis of Circadian Proteins

Cells were lysed in a lysis buffer, as was described [15 (link)]. Samples were run on a 10% SDS polyacrylamide gel and transferred onto nitrocellulose membranes, as was described [15 (link)]. Blots were incubated with antibodies against AMP-activated protein kinase (AMPK), phosphorylated AMPK (pAMPK), BMAL1, phosphorylated BMAL1 (pBMAL1), S6, phosphorylated S6, FAS (Cell Signaling Technology, Beverly, MA, USA), mTOR, CRY1, and CLOCK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and after several washes, with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA). Anti-mouse antibody (Santa Cruz Biotechnologies) was used to detect actin, the loading control. The immune reaction was detected by enhanced chemiluminescence (Santa Cruz Biotechnologies). Finally, bands were quantified by scanning and densitometry and expressed as arbitrary units.