Fig. 4

Proteomics analysis indicated AHNAK is a ubiquitination substrate of RNF38. a. SILAC-based proteomics data of HepG2-vector and HepG2-RNF38 cells. Red plots represent up-regulated proteins and green plots represent down-regulated proteins. b. Histogram showed the total number of up-regulated proteins and down-regulated proteins in two groups. c. Randomly chose eight proteins to verify the proteomics data. d. GO and KEGG analysis were performed for those up or down regulated proteins. e. Venn chart showed the number of substrates of RNF38 between HCCLM3-NC and HepG2-RNF38, and 8 overlapped proteins were included in the diagram. Eight overlapped proteins are listed in the Table. f. Co-IP and western blot were used to definite the RNF38 interacted with AHNAK. g. Western blot revealed that RNF38 expression affected the level of AHNAK protein. h. qRT-PCR showed that RNF38 had no influence on the AHNAK mRNA. i. Immunofluorescent staining images showed that the high level of RNF38 (green) was negatively related to the AHNAK (red) protein level. Nucleus (blue). j. AHNAK were immunoprecipitated from HCCLM3-NC and HCCLM3-shRNA2 and analyzed by ubiquitin antibody; NC negative control, KD knockdown. k. AHNAK’s half-life was prolonged after RNF38 de-regulation. CHX treated time point is 0 h, 2 h, 4 h, 6 h, respectively. And relative AHNAK expression was shown in the right panel. Abbreviations: CHX, cycloheximide; DAPI, 4′, 6-diamidino-2-phenylindole. Scale bars 50 um