Enhanced western lightening chemiluminescence

Briefly, shCon and shRB cells treated with PD 0332991 (500 nM) for three weeks and were harvested by trypsinization, and cell lysis was carried out in radio-immunoprecipitation assay (RIPA) buffer [(150 mmol/L NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH, 8.0)] supplemented with protease inhibitors, phosphatase inhibitors, and phenyl methylsulfonyl fluoride. After sonication, lysates were clarified, and protein concentrations were determined using Bio-Rad Protein Assay Reagent. Protein was subjected to SDS-PAGE and transferred onto Immobilin-P PVDF transfer membranes (Millipore Corp).
The membranes were immunoblotted for RB (BD Sciences, USA), phospho-RB (phospho-serine 780), PCNA, CDK4, CDK6, CyclinA, Caspase3, Cleaved caspase3, SMAC, FOXM1, Survivin/BIRC5, LaminB and GAPDH (Santa Cruz Inc., USA), p16 antibody from Proteintech (USA), Annexin V from GeneTex (Irvine, CA, USA). Protein signals were visualized via X-ray film using enhanced Western lightening chemiluminescence (Perkin-Elmer Life Sciences) and normalized with LaminB or GAPDH loading control.

For protein analysis, cells were harvested by trypsinization, and cell lysis was performed. The membranes were immunoblotted for pRB (BD Biosciences, San Jose, CA), actin, LaminB, p21, NFκB p50, NFκB p65, IκBα cleaved caspase 3 (Santa Cruz Biotechnology Inc, Dallas, TX and Abcam, Cambridge, MA), PLK3 (Sigma-Aldrich, St. Louis, MO), DNA ligase IV (Abcam, Cambridge, MA), CDC25A, CDK2 and p53 (Santa Cruz Biotechnology Inc, Dallas, TX) by standard techniques and visualized using enhanced Western lightening chemiluminescence (Perkin-Elmer Life Sciences, Waltham, MA).