Figure 8.

MARCH5 ubiquitin ligase activity is required for PMP70 ubiquitination and degradation with Torin1 treatment. (A) MARCH5 KO cells were transfected with empty vector (EV), MARCH5-Myc, or catalytically inactive MARCH5-CS-Myc plasmid for 24 h and treated with Torin1 (1 μM for 24 h), as indicated. The cell lysates were immunoblotted with indicated antibodies. (B) The protein levels in A were quantified with ImageJ, then normalized to β-actin. Values are mean ± SD, n = 3 independent experiments. *, P < 0.05; **, P < 0.01; ns, P > 0.05 by two-tailed Student’s t test. (C) WT and MARCH5 KO HeLa cells were transfected with HA-Ub for 24 h and then treated with Torin1 (1 μM for 24 h) and MG132 (10 μM for 4 h). Cell lysates were then subjected to a ubiquitination assay and immunoprecipitated with anti-PMP70 antibody, followed by WB analysis. (D) MARCH5 KO cells were transfected with MARCH5-Myc or catalytically inactive MARCH5-CS-Myc and HA-Ub plasmid for 24 h and treated with Torin1 and MG132, as indicated. The ubiquitination assay was performed, and cell lysates were immunoprecipitated with anti-PMP70 antibody, followed by WB analysis.