Figure 4.

USP8 associates with OGT. A) Mass spectrometry assay of USP8‐associated proteins in LM3 cells was performed, and the specific interactive information between USP8 and OGT was shown. B) Co‐IP assay reveals an association between endogenous USP8 and OGT in LM3 cells. LM3 cells were harvested with RIPA lysis buffer. Co‐IP was performed using antibody as indicated. C) LM3 cells transfected with Myc‐OGT were lysed and the lysates were incubated with GST‐USP8 or GST protein. The interacted OGT was detected by western blot. D) An immunofluorescence assay demonstrated that USP8 and OGT at least partially colocalized in LM3 cells. E,F). USP8 and OGT domain structure and deletion mutants used in the study. J) The USP domain of USP8 interacted with OGT. HEK293 cells were transfected with 2 µg Myc‐OGT together with Flag‐USP8 full‐length or mutants. After 24 h, cells were harvested with NP‐40 lysis buffer. Co‐IP was performed using Myc antibody. The possible interacted USP8 domains were detected by Flag antibody. K) The GT domain of OGT interacted with USP8. HEK293 cells were transfected with 2 µg Flag‐USP8 together with Myc‐OGT full‐length or mutants. After 24 h, cells were harvested with NP‐40 lysis buffer. Co‐IP was performed using Flag antibody. The possible interacted OGT domains were detected by Myc antibody.