Figure 4.

Knocking down Ube2D1 decreases March-I ubiquitination and increases March-I expression. HeLa cells were transfected with control shRNA or Ube2D1 shRNA for 2 days. Cells were then transfected with V5-tagged March-I, and 18 h later, cells were harvested. The expression of Ube2D1, actin, March-I, and zsGreen1 was analyzed by immunoblot of cell lysates. A, a representative blot of three independent experiments. B, the expression of March-I (normalized to zsGreen1) was determined by quantitative densitometry and was expressed relative to that in the control shRNA sample. Error bars represent ± S.D. obtained from three different experiments (each experimental value is indicated with a filled circle). *, p < 0.05. C, lysates of cells transfected as described above were incubated with anti-V5-agarose beads to immunoprecipitate March-I, and the extent of March-I ubiquitination was determined by anti-ubiquitin immunoblotting. The amount of sample loaded was corrected to allow visualization of the extent of March-I ubiquitination for identical amounts of total March-I (determined by anti-V5 immunoblot). A representative blot of three independent experiments is shown. D, the extent of March-I ubiquitination present in each sample was determined by quantitative densitometry and was expressed relative to that in the control shRNA sample. Error bars represent ± S.D. obtained from three different experiments (each experimental value is indicated with a filled circle). *, p < 0.05.