Fig 1: UBTD1 controls ASAH1 ubiquitination to promote EGFR self-phosphorylation.(A–D) DU145 cells were transfected for 48 hr with the indicated siRNA (siCTRLpool or siUBTD1pool or siUBTD1single1 or single2 or siASAH1 single1 or single2). (A) Immunoblot and quantification of ceramide synthase 2 (CerS2) and the lysosomal ceramidase (ASAH1). (B) Immunoblots (up) and quantification (down) of ASAH1 levels in cells treated with cycloheximide (CHX) at different time points. Immunoblot of UBTD1 shows the level of siRNA depletion. CHC was used as a loading control. (C) Immunoblots show ASAH1 ubiquitylation in HEK cells in different experimental conditions. Cells were transfected, as indicated, with expression vectors for histidine-tagged ubiquitin (His-Ub) together with control siRNA or UBTD1 siRNA. His-Ub crosslinked forms of ASAH1 were purified (IP: His) and the immunoblot of ASAH1 showed ASAH1 ubiquitylation. The immunoblot of ASAH1 (bottom panel) was performed in parallel to verify the amounts of ASAH1 protein engaged in His-Ub purifications. The immunoblot of UBTD1 shows the level of siRNA depletion. (D) Immunoblot and quantification of p-EGFR (Y1068 or Y1086) and p-STAT3. p-STAT3, p-ERK and p-AKT levels were quantified by calculating the ratio between phospho-protein and total-protein, both normalized to loading control signal. Immunoblot of UBTD1 shows the level of siRNA depletion. n ≥ 3 independent experiments; ns = non-significant, **p<0.01, ***p<0.001; ****p<0.0001; (A,D) two-way ANOVA and Bonferroni’s multiple comparisons test; data are mean ± s.e.m.Figure 3—source data 1.Uncropped western blot for Figure 3.Figure 3—source data 2.Row data for Figure 3.