Figure 5.

AMBRA1 ubiquitinates ALDH1B1 by mediating TRAF6, and ALDH1B1 is ubiquitinated by K27-, K29-, and K33-linked ubiquitin chains. (A) Ubiquitination of Myc-ALDH1B1 in AMBRA1 WT-, Δ43-, and AA-overexpressing cells as identified by Co-IP. Empty vector and Flag-AMBRA1 WT, Δ43, and AA were cotransfected with HA-Ub and Myc-ALDH1B1 into HCT116 cells. After treatment with 10 μM MG-132 for 6 h, immunoprecipitation and western blot analysis were performed (n = 3). (B) Using HA-Ub mutants (HA-Ub K0, K6, K11, K27, K29, K33, K48, and K63), the ubiquitination linkage types of Myc-ALDH1B1 were investigated by a cell-based ubiquitination assay (n = 3).