FIGURE 2.

MARCH-induced down-regulation of HA-DRB-K225R. Mel JuSo cells, stably expressing various HA-tagged DRα and DRβ constructs, were transiently transfected with MARCH I, MARCH VIII, c-MIRwt, and c-MIRmt, and surface HA expression was assessed by FACS. Dot plots show GFP (FL1) on the x axis and PE-HA (FL2) on the y axis. The R3 gate, which represents cells expressing the E3 ligase, as assessed by GFP expression, was set against IgG control at 0.1%. The IgG control was set using cells expressing GFP (R3), to enable direct comparison between with E3 ligase-expressing transfectants. Data are representative of at least three independent experiments. A, expression of MARCH I, MARCH VIII, and c-MIRwt induced down-regulation of surface HA-DRB, HA-DRB-K225R, HA-DRB-L235A,L236A, and HA-DRB-Δ₂₂₅. No reduction in surface expression was observed in cells transfected with c-MIRmt. When compared with IgG control antibody staining, MARCH-induced down-regulation of HA-DRB-K225R and HA-DRB-Δ₂₂₅ was substantial, but less than observed for HA-DRB and HA-DRB-L235A,L236A. B, MARCH I, MARCH VIII, and c-MIRwt induced down-regulation of surface HA-DRA and DRA-K219R. No reduction in surface expression was observed in cells transfected with c-MIRmt. Data are representative of at least three experiments.