CLOCK Protein Expression Analysis

Heart samples from each group at one month were homogenized and lysates at light phase or at different time point were prepared as previously described. H9c2 cells were homogenized immediately after 48 h of transfection with siRNA targeting CLOCK^{5 (link)}. Equal amounts of protein were subjected to CriterionTGX (Tris–Glycine extended) Stain-Free PAGE gels (BioRad) and activated using a ChemiDoc MP Visualization System (BioRad). The protein was then transferred to PVDF membranes as previously described^{49 (link),56 (link)}. Those membranes were then imaged to obtain total protein loadings. The membranes were blocked and probed with the targeted primary antibodies, followed by incubation with secondary antibodies. The protein signal was detected using an ECL Prime Western Blotting System (Cytiva). Protein expression was analyzed by measuring band intensities using Image J software^{49 (link)}. Average photoperiod quantification of CLOCK protein expression was normalized to total protein. The antibodies are described in the Supplemental Materials.