Overexpression of the C-terminal p230 GRIP domain disrupts the intracellular localization of both CD99 and HLA class I and induces down regulation of HLA class I at the cell surface. (A) Wild-type Jurkat T cells that were transiently transfected with plasmid expressing GFP-p230-C230aa were analyzed 72 hours later by IFM. GFP-p230-C230aa fusion protein is efficiently Golgi localized. (B-F) Cells were stained for endogenous CD99, HLA class I, TGN46, GM130, and LAMP1 with, respectively, the CD99 “O662” and the “W6/32” mAbs, a sheep anti-TGN46 antibody, the “35” anti-GM130 and anti-LAMP1“25CD107a” mAbs followed by appropriate secondary Texas Red–conjugated antibodies. (G) Wild-type Jurkat T cells that were stably transfected with a His-tagged plasmid expressing p230-C230aa were analyzed for HLA class I expression using the “W632” mAb; CD47 “B6H12” mAb was used as control. The bottom panel shows the expression level of His-tagged p230-C230aa protein compared with control cells using the anti–His G mAb after 20 minutes permeabilization in methanol.