Figure 5

Stabilization of Snail by USP47. (a) Cells were exposed to normoxia or hypoxia for 72, and mRNA and protein expression levels of Snail in DLD-1 cells were examined RT-PCR (upper left panel)/quantitative real-time PCR (upper right panel) and Western blot analyses, respectively. (b) mRNA and protein expression levels of Snail in siUSP47-transfected DLD-1 cells under normoxic and hypoxic conditions were measured by PCR and Western blot analyses, respectively. (c) Cellular lysates of DLD-1 cells were immunoprecipitated with anti-Snail and anti-USP47 antibodies, and immunoprecipitates were immunoblotted with anti-Snail antibody. (d) DLD-1 cells were stained for USP47 and Snail by immunocytochemical analysis. Nuclei were stained using DAPI. Scale bar = 100 μm. (e) Levels of Snail-attached ubiquitins in USP47-silenced DLD-1 cells were evaluated by immunoprecipitation analysis. (f) Evaluation of ubiquitin-flag construct-transfected control and USP47-deficient DLD-1 cells. Snail immunoprecipitates were immunoblotted with anti-flag antibody. Results in (a,c,e) are representative of three independent experiments. Statistical significance between the groups was assessed by using two-sided student’s t-test; *P < 0.05 and **P < 0.01.