Immobilin p pvdf transfer membranes

For protein analysis, RB-proficient and -deficient cells (LNCaP, PC3, LAPC4, C4-2, PC3-ML), RHAMM-overexpressing cells (PC3, LNCaP, PC3-ML), and RHAMM knockdown cells (PC3, PC3-ML, LNCaP) were utilized. Briefly, the cells were harvested by trypsinization, and cell lysis was carried out in 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 phenylmethylsulfonyl fluoride. After brief sonication, lysates were clarified, protein concentrations were determined using Bio-Rad Protein Assay Reagent, and an equal amount of protein was subjected to SDS-PAGE and transferred onto Immobilin-P PVDF transfer membranes (Millipore). The membranes were immunoblotted for RB (BD Biosciences), phospho-RB (phospho-serine 780), RHAMM, GAPDH, F-actin (phalloidin, Invitrogen Inc), cofilin, phospho-cofilin (S3) (Cell Signaling Technology), ROCK II, F-actin, lamin B, E-cadherin, vimentin, E2F1, E2F2, and RNRII (Santa Cruz Biotechnology and Abcam) by standard techniques and visualized using Enhanced Western Lightning Chemiluminescence (Perkin-Elmer Life Sciences). The signals were normalized with the internal control lamin B or GAPDH.

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.