Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer

Figure 2

(a) RKO cells stable-transfected with pcDNA (IRS-4) or pcDNA were untreated or treated with wortmannin (200 nM) during 18 h and several proteins were analysed by western blot. (b) RKO cells were starved overnight with serum-free medium and stimulated with IGF-1 (25 nM) during 30 min, then harvested, and lysed. Immunoprecipitation with anti-IRS-4 antibody was carried out and tyrosine phosphorylation was analysed by immunoblot. (c) Immunoprecipitations with anti-IRS-4 antibody and (d) anti-IRS-1 antibody was performed in RKO cells after IGF-1 treatment (25 nM) during 30 min, then the association with αp85 was studied by immunoblot. (e) RKO cells with or without IGF-1 stimulation (25 nM) during 30 min were lysed and immunoprecipitated with anti-BRK antibody and its interaction with IRS-4 was studied by western blot. (f) RKO cells stable-transfected with pcDNA (IRS-4) or pcDNA and treated with wortmannin (200 nM) during 18 h were immunoprecipitated with anti-BRK antibody and its association with IRS-4, IGF-1 receptor and phosphorylated IGF-1 receptor were studied by western blot. Negative control (C-) was assessed replacing the lysis extract for sample buffer. Results shown are representative for two or three independent experiments. H.C. = heavy chain of the immunoprecipitation antibody. SB = sample buffer.

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