FIGURE 8.

Gadd45a regulates the association between STAT3 and mTOR. a, a GST pull-down assay was performed with GST-Gadd45a and cell lysates from HeLa cells. A GST tag alone was used as an internal control. The binding proteins were analyzed with mTOR and GST antibodies. b, extracted whole proteins from HeLa cells were immunoprecipitated with mTOR antibody. The precipitates were analyzed by immunoblot analyses. HeLa cells were harvested 36 h after transient transfection with myc-Gadd45a or control vector and prepared for immunoprecipitation with myc (c) and mTOR (d) antibodies (Ab). The precipitated complexes were analyzed with immunoblot analyses. Myc-Gadd45a was detected with myc antibody. e, HeLa cells were transiently transfected with gradually increased doses of myc-Gadd45a vector. 36 h after transfection, cells were collected and prepared for immunoprecipitation (IP) with mTOR antibody. Immunocomplexes were examined by immunoblot analysis. f, HeLa cells were harvested 48 h after transient transfection of siRNA against Gadd45a. Whole protein extracts were prepared for immunoprecipitation with mTOR antibody. Immunoblot analysis was used to detected immunocomplexes. NC, negative control. g, Gadd45a^+/+ and Gadd45a^−/− MEFs were harvested for immunoprecipitation with mTOR antibody. The precipitated complexes were analyzed as in f. Non-specified IgG was used as an internal control. β-actin was used as the loading control.