Figure 5.

Knocking down Ube2D1 enhances March-I-dependent down-regulation of MHC-II. HeLa-CIITA cells were transfected with control siRNA or Ube2D1 siRNA for 2 days. Cells were then transfected with an expression vector encoding wildtype March-I (A and B) or catalytically inactive March-I I65A/W97A mutant (C and D) in the V5-tagged March-I/IRES/zsGreen1 vector. After 18 h, cells were harvested and examined by FACS analysis for expression of V5-tagged March-I, MHC-II, and zsGreen1. A and C, the extent of March-I up-regulation in the Ube2D1 siRNA sample was expressed relative to the expression of March-I present in the control siRNA sample (normalized to 1.0 for each independent experiment). A representative FACS plot is shown, and error bars represent ± S.D. obtained from three different experiments. *, p < 0.05. B and D, MHC-II expression on siRNA-treated HeLa-CIITA cells transfected with empty zsGreen1 vector (no March-I) or V5-tagged wildtype March-I (B) or V5-tagged March-I I65A/W97A mutant (D) was determined by FACS analysis. B, the extent of MHC-II down-regulation by wildtype March-I in the Ube2D1 siRNA sample was expressed relative to the expression of MHC-II present in the control siRNA sample (normalized to 1.0 for each independent experiment). D, the expression of MHC-II in cells expressing V5-tagged March-I I65A/W97A with either control siRNA or Ube2D1 siRNA was expressed relative to the expression of MHC-II in control-transfected cells (normalized to 1.0 for each independent experiment). A representative FACS plot is shown, and error bars represent ± S.D. obtained from at least three different experiments (each experimental value is indicated with a filled circle). *, p < 0.05; ns, not significant.