Fig. 5

TRIM47 upregulates the expression of CCL15 and CCR1 via interacting with SMAD4 and enhancing ubiquitylation and degradation of SMAD4. a and b Western blot was performed in HCT116 and HT29 cells transfected with control siRNA and TRIM47 siRNAs (n = 3). c Western blot was performed in SW480 cells transfected with vector plasmids and TRIM47 overexpression plasmids (n = 3). d Western blot was performed in SW480 cells transfected with TRIM47 overexpression plasmids and treated with DMSO or MG132 (n = 3). e Western blot was performed in HCT116 cells transfected with TRIM47 siRNAs and treated with DMSO or MG132 (n = 3). f The amount of ubiquitin that co-immunoprecipitated with SMAD4 in SW480 cells transfected with TRIM47 overexpression plasmids. Western blot data of TRIM47, ubiquitin and GAPDH from 20% input (left). Anti-ubiquitin and anti-SMAD4 antibody were used for western blot to determine the ubiquitination level of SMAD4 (right) (n = 3). g The amount of ubiquitin that co-immunoprecipitated with SMAD4 in HCT116 cells transfected with TRIM47 siRNAs (n = 3). h The amount of ubiquitin that co-immunoprecipitated with SMAD4 in SW480 cells transfected with TRIM47 overexpression plasmids and the △RING domain plasmids (n = 3). RING, RING-finger domain; PR, proline-rich region; B-box, B-box-type zinc finger; CC, coiled coil region; SPRY, C-terminal SplA/ryanodine receptor domain; Flag-, Flag-tagged; △RING, deletion of the RING domain. i Co-immunoprecipitation detected the interaction of TRIM47 and SMAD4 in SW480 and HCT116 cells. The specific immunoprecipitation of TRIM47 and SMAD4 was confirmed by western blot (n = 3). j Immunofluorescence revealed that TRIM47 is co-localized with SMAD4 (n = 3)