Fig. 5. CRBN promotes the ubiquitination of TRIM47.
A HEK293T cells were transfected with FLAG-TRIM47 for 48 h. Cell extracts were subjected to immunoprecipitated with anti-FLAG antibody or a control IgG. CRBN was identified via mass spectrometry. B HEK293T cells were co-transfected with FLAG-TRIM47 and MYC-CRBN for 48 h. Cell extracts were immunoprecipitated with anti-FLAG or anti-MYC antibodies. FLAG-TRIM47 and MYC-CRBN were detected by western blot. C Western blot analysis of TRIM47 protein levels in SMMC7721 cells transfected with MYC-CRBN for 48 h. D SMMC7721 cells were transfected with MYC-CRBN plasmid for 48 h and treated with MG132 (10 μM) for another 4 h. Western blot analysis of TRIM47 protein levels. E SMMC7721 cells were co-transfected with MYC-CRBN and HA-ubiquitin plasmids for 48 h. Cell extracts were immunoprecipitated with anti-TRIM47 antibody. The immunoprecipitates were detected with anti-ubiquitin and anti-TRIM47 antibodies. F Immunoprecipitation analysis of TRIM47 ubiquitination in SMMC7721 cells co-transfected with MYC-CRBN and HA-ubiquitin (K48) or HA-ubiquitin (K63). Cell extracts were immunoprecipitated with anti-TRIM47 antibody. The immunoprecipitates were detected with anti-HA and anti-TRIM47 antibodies. G SMMC7721 cells were treated with HY12759 for 8 h. Total cell extracts were immunoprecipitated with anti- TRIM47 antibody. The immunoprecipitates were detected with anti-ubiquitin, anti-TRIM47 and anti-CRBN antibodies. H Western blot analysis of TRIM47 protein levels in TRIM47 stably knockdown SMMC7721 cells co-transfected with FLAG-TRIM47 or FLAG-TRIM47R210/582K and MYC-CRBN plasmids for 48 h.