Figure 5
Smad7 knockdown prevents PKR-p-58IPK interaction in DLD-1 cells and activates the PKR/CHOP axis in human CRC explants. (a) Total proteins extracted from DLD-1 cells were immunoprecipitated by an anti-human Smad7 or control isotype (ve−) antibody and then subjected to immunoblotting analysis using PKR, p58IPK and Smad7 antibodies. (b) Total proteins extracted from DLD-1 cells transfected with either Smad7 sense (S) or antisense (AS) oligonucleotide (both used at 2 μg/ml) were immunoprecipitated by an anti-human PKR or control isotype (ve−) antibody and then subjected to immunoblotting analysis using p58IPK, Smad7 and PKR antibodies. One of three representative experiments is shown. (c) Freshly obtained CRC explants were cultured in the presence of Smad7 S or AS oligonucleotide (both used at 8 μg/ml) for 24 h. Smad7, p-PKR, PKR, CHOP, procaspase-3 and cleaved caspase-3 protein expression were assessed by western blotting. One of four representative experiments is shown. Right panels: Quantitative analysis of Smad7/β-actin, p-PKR/PKR, CHOP/β-actin and cleaved caspase-3/procaspase-3 protein ratio in extracts of CRC explants measured by densitometry scanning of western blots. Values are expressed in a.u. (arbitrary units) and are the means±S.E.M. of four experiments
