P eif4e s209

Western blotting was performed either with the Simple Western assay by Simon (ProteinSimple, Santa Clara, CA, USA) according to manufacturer instruction [54 (link)] or as previously described [13 ]. Antibodies used were as follows: total RNAPII, phosphorylated RNAPII serine-2 (p-RNAPII^S2) and serine-5 (p-RNAPII^S5) (Covance, NJ, USA), 4E-BP1, p-4E-BP1 (pT⁷⁰), β-Actin, caspase-3, caspase-7, CDK9, eIF4E, p-eIF4E (S²⁰⁹), eIF4G, p-Erk^T202/Y204, p-p38^T180/Y182, p38 MAPK, rpS6, Mcl-1, Mnk1, PARP, cleaved PARP (Cell Signalling Technology, Danvers, MA, USA), Erk (ProteinSimple), MDM-2 (Becton Dickenson, Franklin Lakes, NJ, USA), Bcl-2, cyclin D1, p-S6^S240/S244, p53 (Dako, Glostrap, Denmark). Both anti-mouse and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugated antibodies were obtained from Dako. Enhanced Chemiluminescence (ECL) reagents were obtained from GE Life Sciences.

Lysates were made using lysis buffer consisting of 50 mM Tris HCl at 7.5 pH, 250 mM NaCl, 0.5% (v/v) NP-40, and 5 mM EDTA, with proteases and phosphatases. A total of 50 µg of protein was loaded into SDS-PAGSE gels and transferred onto iBlot 2 nitrocellulose membranes (Invitrogen). The following antibodies were purchased from Cell Signaling Technology: RPSKB1, RPSKB2, RPSKA1, STAT5A, AKT1, p-AKT S473, p-S6, S6, p-eIF4E S209, and eIF4E. β-actin (A5316) was purchased from Sigma.