Fig. 2
EIF3H associates with OGT and enhances its stability. A The immunofluorescence assay presented that EIF3H and OGT at least partially colocalized in LM3 cells. B Co-IP assay showed a correlation between endogenous EIF3H and OGT in LM3 cells. C-D EIF3H and OGT structure domain and deletion mutants were applied in the study. E The JAM/MP domain of EIF3H interacted with OGT. HEK293 cells were transfected with 2 µg Myc-OGT and GFP-EIF3H full-length or mutants. Cells were harvested with NP-40 lysis buffer for 24 h. Co-IP was performed using a GFP antibody and the possible interacted EIF3H domains were detected by Myc antibody. F OGT interacted with EIF3H through its GT domain. HEK293 cells were transfected with 2 µg GFP-EIF3H together with Myc-OGT full-length or mutants., Cells were harvested with NP-40 lysis buffer After 24 h. Co-IP was performed using Myc antibody. The possible interacted OGT domains were detected by GFP antibody. G In the presence of the proteasome inhibitor MG132, the knockdown of EIF3H did not further reduce the OGT protein expression level. LM3 cells were transfected with siControl or siEIF3H. After 48 h, cells were treated with 10 µM MG132/vehicle for 6 h, cell lysates were prepared for Western Blot analysis. H LM3 cells were transfected with EIF3H (wild type or mutant DDQ/AAA) together with EIF3H siRNA. The OGT protein levels were detected by western blot assay. I Silencing EIF3H decreases OGT half-life in LM3 cells. LM3 cells were transfected with siControl or siEIF3H. After 48 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. J The mutant EIF3HDDQ/AAA did not increase OGT half-life in HEK293 cells. HEK293 cells were transfected with Myc-tag, Myc-EIF3H or Myc-EIF3HDDQ/AAA plasmids. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot assay