Flag and gapdh

Western blotting analysis was performed as previously described [19 (link)] with antibodies against flag and GAPDH (Abcam, Cambridge, MA, USA). GAPDH was used as an endogenous control to normalize the protein loading.

Cells were lysed in either a sodium dodecyl sulfate (SDS) lysis buffer: a 1:3 mixture of buffer I (5% SDS, 0.15 M Tris-HCl [pH 6.8], 30% glycerol) and buffer II (25 mM Tris-HCl [pH 8.3], 50 mM NaCl, 0.5% NP-40, 0.1% SDS, 1 mM EDTA) or Urea 8 M, containing 0.5 mM N-ethylmaleimide (NEM), 0.5 mM NaF and 2 mM sodium Na₃VO₄ and protease inhibitors (Sigma). After lysis, an equal amount of protein for each sample was resuspended in denaturing sample loading buffer, separated on SDS-PAGE gel and immunoblotted with the indicated antibodies. The following antibodies were used: SMAD4, CHK1, p-CHK1 and βTRCP1 (Cell Signaling Technology), p63, pH2Ax, Flag and GAPDH (Abcam), HPV16 E7, p53, H3 and Rad51 (Santa Cruz Biotechnology), and Vinculin (Merck Millipore). Membranes were then incubated with the appropriate horseradish peroxidase (HRP) secondary antibodies and the signal was acquired with Chemidoc (Bio-Rad). Densitometric analysis of the intensity of the protein bands were preformed using ImageJ software (Rasband W.S., ImageJ, U. S. National Institutes of Health), and standardized to the housekeeper levels.