Rabbit anti eif4a

Cells were resuspended in lysis buffer (100 mM NaCl, 15 mM MgCl₂, 2 mM EDTA, 20 mM Hepes, 10% Glycerol, 1 mM dithiothreitol, 2 mM Na-ortovanadate, Protease-Inhibitor Cocktail (Sigma-Aldrich) and 1% Triton X-100). After10 min of incubation in ice, extracts were centrifuged for 10 min at 12 000 rpm at 4 °C, supernatants were resuspended in SDS-page sample buffer and boiled for 5 min. Western blot analysis was performed as previously described.^{31 (link)} The following primary antibodies (overnight at4 °C) were used: rabbit anti-Actin (1 : 1000, Sigma-Aldrich), mouse anti-GAPDH (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-E-cadherin (1 : 1000, Santa Cruz Biotechnology), mouse anti-Vimentin (1 : 1000, Santa Cruz Biotechnology), rabbit anti-ZEB1 (1 : 1000, Sigma-Aldrich), rabbit anti-eiF4E (1 : 1000, Cell Signalling Technology, Danvers, MA, USA), rabbit 4EBP1 (1 : 1000, Cell Signalling Technology), rabbit anti PARP-1 (1 : 1000, Cell Signalling Technology), rabbit anti eiF4A (1 : 1000, Abcam, UK), rabbit anti-eIF4G (1 : 1000, Cell Signalling Technology). Secondary anti-mouse or anti-rabbit IgGs conjugated to horseradish peroxidase (Amersham, UK) were incubated for 1 h at RT (1 : 10 000). Immunostained bands were detected by chemiluminescence method (Santa Cruz Biotechnology).

Protein extracts were separated by SDS-PAGE and analysed by Western blot as previously described [38 ]. The primary antibodies used are: rabbit anti-actin (1:1000, Sigma-Aldrich); rabbit anti-4E-BP1 (1:1000), rabbit anti-4EBP1 pSer65 (1:500), rabbit anti-eIF4E (1:1000), rabbit anti-eIF4G (1:1000), rabbit anti-AKT pThr308 (1:200), rabbit anti-rpS6 pSer240-244 (1:1000), rabbit anti-rpS6 pSer235-236 (1:1000) (all from Cell Signalling Technology); rabbit anti-pSer473 AKT (1:1000) (from BioSource); rabbit anti-eIF4A (1:1000) (Abcam). Actin was used as loading control to normalize the samples. Secondary IgGs conjugated with horseradish peroxidise (1:10 000; Amersham Bioscience) were incubated for 1 hour and signals were detected by enhanced chemioluminescence (Santa Cruz Biotechnology).