Phospho eif4e s209

Cells lysis and western blot were carried out as previously described [18 (link)]. Antibodies for: ILK, Akt, E-cadherin, Vimentin, GSK-3β, phospho-GSK-3β (Y216), β-catenin (all Transduction Laboratories BD), AR, Src, phospho-Src (Y416), phospho-ERK (T202/Y204), phospho-Akt (S473), phospho-GSK-3β (S9), D₁, D₃ and phospho-eIF4E (S209) (all Cell Signaling Technology Inc.), N-cadherin (R&D) and β-actin, ZEB1, (all Sigma) SNAIL (ABGENT), Calnexin, HSP-90 (all Calbiochem) were used to detect indicated proteins. The presence of the primary antibody was revealed with horseradish peroxidase-conjugated secondary antibodies diluted 1:2000 (Cell Signaling Technology Inc) and visualized with an enhanced chemiluminescence detection system (Bio-Rad) as previously described [19 (link)]. β-Actin served as a loading control. All immunoblots were stripped with stripping buffer containing 25 mM glycine–HCl, pH 2, 1% (wt/v) SDS for 30 min, and incubated in antibody against β-actin (dilution, 1:3000; Sigma-Aldrich), which served as a loading control. To obtain quantitative results, immunoblots were scanned using the public domain ImageJ software (National Institute of Health). Each data point was normalized against its corresponding actin data point.

Cycloheximide, 5-aza-2′-deoxycytidine (5-Aza), trichostatin A (TSA), and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The indicated primary antibodies against the following proteins were used in this study: STAT1/2/3/4/5/6 (#9172/4594/12640/5097/9363/9362), DNMT1/3a/3b (#5119/3598/67259), cleaved-caspase 3/7 (#9664/9491), cleaved PARP (#5625), S6K (#2708), phospho-S6K (T389) (#9205), S6 (#2217), phospho-S6 (Ser235/236) (#2211), mTOR (#2972), 4E-BP1 (#9644), phospho-4E-BP1 (T70) (#9455), eIF4E (#2067), phospho-eIF4E (S209) (#9741), eIF4G (#2469), Rheb (#13879), TSC2 (#4308), p-TSC2 (T1462) (#3617), p-TSC2 (S1387) (#5584), ICAM1 (#4915), JAK2 (#3230), NFATC2 (#4389) and Myc taq (#2278) (Cell Signaling Technology, Danvers, MA, USA); anti-HIF-1α (610958) (BD Biosciences, San Jose, CA, USA); and anti-actin (sc-1616) and GAPDH (sc-48,167) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies used were anti-goat IgG HRP (81–1620; Invitrogen, Carlsbad, CA, USA), anti-mouse IgG HRP (G-21040; Invitrogen), and anti-rabbit IgG HRP (111–035-003; Jackson Laboratories, Bar Harbor, ME, USA).