Immunoblotting Analysis of Cell Cycle Proteins

Immunoblots were performed as previously described^{69 (link)}. Briefly, membranes were probed with the following primary antibodies: Cyclin D1 (1:1000; Cell Signaling Technology), Cyclin E (1:1000, EDM Millipore Temecula, CA, USA), Cdk1/2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (1:1000 Cell Signaling Technology, Danvers, MA, USA), p21/WAF1/Cip1 clone CP74 (1:1000, EDM Millipore Temecula, CA, USA), MitoProfile Total OXPHOS Human WB Antibody cocktail (1:500; Abcam Cambridge, UK) and β-Actin (1:5000; Sigma Aldrich, St. Louis, MO, USA). The secondary antibody was horseradish peroxidase-conjugated (1:2500; Cell Signaling Technology) and the signals developed by enhanced chemiluminescence kit (Clarity Western ECL Substrate, Bio-Rad), acquired by ChemiDoc Imaging System XRS + (BioRad) and analyzed for densitometry with the ImageJ Lab 4.1 software.