Anti t202 y204 phosphorylated erk1 2

Immunoblot analysis was performed using the following antibodies; anti–T²⁰²/Y²⁰⁴-phosphorylated ERK1/2, anti-ERK1/2, anti-T³⁸⁹-phosphorylated p70S6K, anti-p70S6K, anti-S⁵¹-phosphorylated eIF2α, anti-eIF2α (all Cell Signaling, Herts, UK), anti-MYC (9E10; Calbiochem, Nottingham, UK) anti-β-actin (Sigma Chemicals). Analysis used equal protein loading following quantitation of protein content using the BioRad Protein Assay (BioRad, Hemel Hempstead, UK). Secondary antibodies were HRP-conjugated rabbit, mouse or goat antibodies (Dako, Cambridgeshire, UK) and images were captured using the ChemiDoc-It Imaging System with a BioChemi HR camera (UVP, Cambridge, UK). Immunoblot signals were quantified using ImageJ (http://imagej.nih.gov/ij/). For quantitation of phosphorylation, expression values were normalized to the loading control (β-actin) and then the relevant total protein. For quantitation of MYC, expression values were normalized to β-actin.

SDS–PAGE was performed using equal protein loading following quantitation of protein content using the BioRad Protein Assay (BioRad, Hemel Hempstead, UK). Immunoblot analysis was performed using the following antibodies; anti-T²⁰²/Y²⁰⁴-phosphorylated ERK1/2, anti-ERK1/2, (both Cell Signaling Technology, Hitchin, UK), anti-MYC (9E10; Calbiochem), anti-MCL-1 (Santa Cruz Biotechnology), anti-GAPDH (Cell Signaling Technologies) and anti-HSC70 (Santa Cruz, Heidelberg, Germany). Secondary antibodies were HRP-conjugated rabbit, mouse or goat antibodies (GE Healthcare, Amersham, UK) and images were captured using the ChemiDoc-It Imaging System with a BioChemi HR camera (UVP, Cambridge, UK). Immunoblot signals were quantified using ImageJ (http://imagej.nih.gov/ij/). Expression of phospho-ERK1/2 was normalized to total ERK1/2 expression, whereas expression of MYC and MCL1 were normalized to loading controls (GAPDH or HSC70).

SDS-PAGE was performed using equal protein loading following quantitation of protein content using the BioRad Protein Assay (BioRad, Hemel Hempstead, UK). Immunoblot analysis was performed using the following antibodies; anti-PDCD4 (Cell Signaling Technology, Danvers, US), anti-MYC (9E10; Merck Life Science UK, Gillingham, UK), anti-ubiquitin (P4D1; Santa Cruz Biotechnology, Dallas, US), anti-ERK1/2, anti–T²⁰²/Y²⁰⁴-phosphorylated ERK1/2, anti-p90RSK, anti-S³⁸⁰-phosphorylated p90RSK, anti-p70S6K, anti-T³⁸⁹-phosphorylated p70S6K, (all from Cell Signaling Technology), anti-β-actin (Cell Signaling Technology) and anti-HSC70 (Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase-conjugated rabbit or mouse antibodies (GE Healthcare, Amersham, UK) and images were captured using the ChemiDoc-It Imaging System with a BioChemi HR camera (UVP, Cambridge, UK). Immunoblot signals were quantified using ImageJ (http://imagej.nih.gov/ij/). Expression of phospho-proteins were normalized to their corresponding total protein expression, whereas expression of all other proteins were normalized to a loading control.