Fig. 4: Nedd4-1(NE) binds VPS33B & VPS16B and ubiquitinates VPS16B.

HEK293T cells were transfected with the indicated constructs. Cell lysates were (A–C) subjected to Flag immunoprecipitation (IP), or (D, E, G) incubated in 1% SDS and boiled to dissociate non-covalent protein complexes prior to Flag IP, and the indicated proteins were detected by immunoblotting (Ub: ubiquitin). The ubiquitination signal was normalized to both VPS16B (or VPS33B) IP levels and Nedd4-1(NE) (or Nedd4-1) lysate levels. F Protein levels of Flag-VPS16B were quantified and normalized to cellular actin levels. In (A), Flag blots were imaged at the same time and images were corrected using the same parameters (MW: molecular weight). Constructs labeled “CS” correspond to full-length, catalytically inactive (Cys → Ser) mutants, and the construct labeled E70Q in (G) represents the lymphangiectasia patient mutation. All data are means ± SD of (A–E) 3 or (F, G) 4 independent experiments. Statistical significance was determined using (A–C) Student’s t-test, or (D–G) one-way ANOVA (N.S.: not significant; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001). The markers beside each blot indicate molecular weight (measured in kDa).