FIGURE 8.

Ubiquitination of MHC class II in the presence and absence of Ii. To determine if ubiquitination was influenced by the presence of Ii, 293T cells were transiently co-transfected with Ii and wild-type or mutated forms of HLA-DRα and HLA-DRβ, together with either MARCH I or MARCH VIII. A, FACS analysis of 293T cells transfected with DRA/DRB and Ii demonstrates that the majority of transfected cells express both class II and Ii. B, 293T cells were transiently co-transfected with Ii and wild-type or mutated forms of DRα and DRβ, together with either MARCH I or MARCH VIII. FACS profiles of surface L243-reactive class II expression demonstrate that all combinations of DRα and DRβ are down-regulated by MARCH I and MARCH VIII, except for DRA-K219R/DRB-K225R, as previously seen in the absence of Ii (Fig. 3). C, HLA-DR was immunoprecipitated (IP) from lysates of the transfected cells depicted above, using L243 monoclonal antibody directly conjugated to Sepharose. After standard SDS-PAGE separation and Western transfer (WB), DRα was detected with HRP-TAL1B5 (bottom), and ubiquitinated HLA-DR was detected with the anti-ubiquitin antibody HRP-P4D1 (top). Lanes 1–4 were transfected with MARCH I, and lanes 5–8 were transfected with MARCH VIII. Lanes 1 and 5 expressed DRA and DRB; lanes 2 and 6 expressed DRA and DRB-K225R; lanes 3 and 7 expressed DRA-K219R and DRB; and lanes 4 and 8 expressed DRA-K219R and DRB-K225R. Comparison with Fig. 3C shows that the pattern of ubiquitination of DRα and DRβ is similar in the presence or absence of Ii. Data are representative of two independent experiments.