E2f1 sc 193

Cells were harvested in lysis buffer containing 62.5 mM Tris-HCl (pH 6.8)/2% SDS, and protease and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA, P8340, P5726, P0044). Protein concentrations were measured using the bicinchoninic acid reagent (Pierce, Waltham, MA, USA, 23228, 1859078). Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane filter (Bio-Rad, Hercules, CA, USA, 1620177). After transfer, membranes were blocked at 25 °C for 1 h in buffer containing 0.1 M Tris (pH 7.4), 0.9% NaCl, 0.05% Tween 20, and 5% nonfat dry milk.
Membranes were then incubated with the appropriate antibodies overnight at 4 °C, at the dilutions indicated as follows: poly(ADP-ribose) polymerase (PARP, #9542), 1:1000; cleaved lamin A (#2035), 1:2000; cytochrome c oxidase subunit IV (COX IV, #4850), 1:2000 (Cell Signaling, Danvers, MA, USA); β-actin A2228) 1:5000 (Sigma, St. Louis MO, USA, A1978); E2F-1 (sc-193), 1:1000; RET (sc-167), 1:1000; mortalin (sc-133137), 1:5000; p27^KIP1 (sc-1641) 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). SuperSignal West Pico and Femto chemiluminescence kits (Pierce, Waltham, MA, USA, 34579 and 34094) were used for visualization of the signal. For densitometry, immunoblots were analyzed using Image Lab software (Bio-Rad, Hercules, CA, USA).