Fig. 4

TRIB2 interacts with AP4 and regulates the function of AP4. a co--IP analysis with anti-TRIB2 or anti-AP4 antibody in SW48 cells. b co-IP analysis with anti-Flag or anti-His antibody in HEK 293 T cells. HEK 293 T cells were transfected with Flag-AP4 and His-TRIB2. After 48 h of transfection, whole cell lysates were immunoprecipitated with an anti-Flag or anti-His antibody and blotted with an anti-His or anti-Flag antibody, respectively. c In vitro GST pull down assay to verify the binding of TRIB2 and AP4. Purified His-AP4 protein was incubated with GST or GST-TRIB2 bound to glutathione-Sepharose beads at 4 °C, and the interaction was confirmed by western blot (up panel); the expression of GST and GST-TRIB2 was confirmed by coomassie blue staining (down panel). d and e Mapping of TRIB2 regions binding to AP4. Flag-AP4 was co-transfected with TRIB2-deleption mutants into HEK293T cells. After 48 h of transfection, the whole cell lysates were immunoprecipitated with anti-Flag antibody and blotted with an anti-His antibody. f luciferase activities of p21 promoter in SW48 cells transiently transfected with p21-luc, Flag-AP4 and His-TRIB2 or its truncated mutants, as indicated. g and h ChIP analysis of AP4 and TRIB2 binding at the p21 promoter. SW48 cells were transfected with Flag-AP4 or His-TRIB2. After 48 h of transfection, the chromatin complexes from transfected SW48 cells were subjected to immunoprecipitate with Flag or His antibody overnight for ChIP assay. i ChIP analysis of AP4 binding at the Amp (− 270) region of p21 promoter in TRIB2 knockdown or overexpressed SW48 cells. Results are presented as mean ± SD from three independent assays, * p < 0.05, ** p < 0.01, *** p < 0.001, t-test