Protein Extraction and Western Blot Analysis

Whole cell extracts of transfected HEK293 cells lysed on ice with cell lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (vol/vol) Nonidet P-40% and 0.5% deoxycholic acid containing Complete Protease Inhibitors (Roche) and PhosStop Phosphatase Inhibitors (Roche)) were analyzed by denaturing SDS-PAGE gel, which was transferred on PVDF membrane (Immobilon, Millipore). The blot was probed using the indicated primary antibodies: anti-myc (9E10) (sc-40, Santa Cruz Biotechnology; RRID:AB_627268) and anti-tubulin (ab52623, Abcam; RRID: AB_869991) were purchased from commercial providers, while rabbit polyclonal antibodies were generated against phospho-Ser478, phospho-Ser659 or phospho-Ser662 of mouse PER2 and purified against the phosphopeptides by Abfrontier (Young In Frontier Co.). The phosphopeptides for phospho-Ser478 and phospho-Ser659 have been described elsewhere (Narasimamurthy et al., 2018 (link); Zhou et al., 2015 (link)); the phosphopeptide KAESVVpSLTSQ-Cys was used to generate the phospho-Ser662 antibody. HRP-conjugated goat secondary antibodies for anti-rabbit (1706515, Bio-Rad) and anti-mouse (1706516, Bio-Rad) were with standard ECL reagents (Thermo Fisher Scientific). Densitometric analysis of western blot bands was performed using ImageJ software (National Institutes of Health).