Western Blot Analysis of Cellular Proteins

Cell lysates were quantified with the Bio-Rad protein assay kit II (Bio-Rad Laboratories, 500-0002EDU). The boiled lysates were resolved by SDS-PAGE, transferred to PVDF membranes and probed with the primary antibodies for various time, The membranes were washed with TBS-T (0.01 M TRIS-HCl Buffer, 8.8 g/L NaCl, 0.1% Tween-20), then incubated with suitable HRP-conjugated second antibodies (Dako, P016102 and P021702). Protein bands were visualized using standard chemical luminescence methodology.
The sources of primary antibodies were as follows: SAG monoclonal Ab was raised against the RING domain (AA44-113)50 (link), cullin1 (Santa Cruz Biotechnology, CA, USA), RBX1, caspase-3, PARP, ORC1, CDT1, p16, p21, p27, BIM, cyclin B1,E-Cadherin, N-Cadherin, Vimentin, GAPDH, phospho-mTOR (Ser2448), phospho-Akt (Ser473), phospho-CHK1 (Ser345), phospho-CHK2 (Thr68), phospho-Histone H3 (Ser10), phospho-p70 S6 (Thr389), phospho-4E-BP1 (Thr70), phosphor-IkBα (Ser 32), IkBα and DEPTOR (Cell Signaling Technology, Denver, CO, USA), phospho-H2A.x (Ser139) (Millipore, Bedford, MA, USA), NEDD8, NOXA, PHLPP1 (Abcam, Cambridge, MA), SQSTM1/p62, LC3 (Sigma, St. Louis, MO), phospho-ATM (Ser1981) (Rockland, Limerick, PA), HIF-1α (Novus Biologicals, Littleton, CO, USA).