Paip2

Cells were washed with ice-cold PBS and lysed in ice-cold RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 10 mM Tris–HCl pH 8.0) supplemented with phosphatase and protease inhibitor cocktails (Sigma-Aldrich). The lysates were incubated on ice for 30 min, with occasional vortexing, and centrifuged at 12,000 g for 10 min at 4 °C. The supernatants were transferred and assayed for protein concentration using a Quick Start Bradford protein assay kit (Bio-Rad). Equal amounts of proteins (20–80 μg) were subjected to SDS–PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad). Antibodies to the following proteins were used: PABPC1 (Cell Signaling Technology), LARP1 (Cell Signaling Technology), PAIP1 (Thermo Scientific), PAIP2 (Santa Cruz Biotechnology), RPL29 (Thermo Scientific), PUS7 (Sigma-Aldrich), DHX36 (Protein Tech), FLAG (Sigma-Aldrich) and puromycin (Merck Millipore). Either β-actin (Sigma-Aldrich) or GAPDH (Abcam) were used as a loading control. The antibody dilutions can be found in the Reporting Summary.

Whole-cell lysates were prepared from cell lines with RIPA lysis buffer kit (Santa Cruz Biotechnology, Santa Cruz, CA), and the protein concentrations were quantified using a Bio-Rad protein assay (Bio-Rad, Hercules, CA). Whole-cell proteins (30 μg) were separated on 8% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham Corp., Arlington Heights, IL). The membranes were then probed with antibodies against the proteins of H3F3b, BAG-2, Paip2 (Santa Cruz, Santa Cuz, CA), BMI-1 (Millipore, Temecula, CA), EGFR, Bax, and β-actin (Sigma), respectively, for 24 hours. Moreover, membranes were also probed with specific cell cycle, apoptotic and anti-apoptotic antibodies against the following proteins: cyclin A, cyclin B, cyclin D1, p53, caspase 3, caspase 9, NFkb MDM2, BCL2, and BCLxl (Sigma). Washed blots were then incubated with horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz Biotechnology) for one hour at room temperature. Blots were developed using a peroxidase reaction and visualized with the ECL detection system (Bio-Rad,).