FIGURE 2.

MARCH E3 ligases polyubiquitinate lysine 230 of CD8-DMα. CD8-DMα and CD8-DMα-K230R reporter molecules were co-transfected into HEK-293T cells together with wild type (wt) or mutant (mut) constructs of EGFP-MARCH1, EGFP-MARCH8, EGFP-MARCH9, or pEGFP. After 24 h, CD8-DMα was immunoprecipitated with nondepleting amounts of mAb OKT8 and analyzed by SDS-PAGE and Western blotting (WB). Ubiquitinated products were detected with P4D1-HRP (upper). The lower panel was probed with anti-CD8 as a loading control. Lanes 1–8 were run on a single gel and lanes 9 and 10 on a separate gel. Bands migrating as ubiquitinated CD8-DMα products are indicated. Data are representative of three independent experiments.