Figure 1
Smad7 colocalizes and interacts with eIF2α in colon cancer cells and controls eIF2α downstream signaling. (a) Representative confocal laser scanning microscopy images showing Smad7 and eIF2α colocalization in DLD-1 cell line. Scale bars, 25 μm; scale bar inset, 10 μm. (b) 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 eIF2α and Smad7 antibodies. One of three representative experiments in which similar results were obtained is shown. (c) Representative immunofluorescence pictures of DLD-1 cells showing that Smad7 knockdown enhances eIF2α (Ser-51) phosphorylation. Cells were transfected with either Smad7 sense (S) or antisense (AS) oligonucleotide (both used at 2 μg/ml). After 24 h, cells were cultured for further 6 h, fixed and stained as described in (a). One of three representative experiments is shown. Scale bars, 20 μm. (d and e) Representative immunofluorescence images of DLD-1 cells showing an increase of ATF4 and CHOP expression following Smad7 knockdown. Cells were transfected with either Smad7 S or AS (both used at 2 μg/ml). After 24 h, cells were cultured for further 12 h, fixed and stained with DAPI nuclear staining (blue), anti-ATF4 or anti-CHOP and secondary Alexa Fluor 546 antibody (red). One of three representative experiments is shown. Scale bars, 20 μm
