Skip to main content
. 2021 Nov 8;22(21):12079. doi: 10.3390/ijms222112079

Figure 4.

Figure 4

AMBRA1, DDB1, and TRAF6 are able to ubiquitinate ALDH1B1. (A) Empty vector (3×F vector) and Flag-tagged AMBRA1 (Flag-AMBRA1) were cotransfected with HA-tagged ubiquitin (HA-Ub) into pCMV-Myc-ALDH1B1 HCT116 cells. Ubiquitination of Myc-ALDH1B1 was analyzed by immunoprecipitating with anti-c-Myc agarose affinity gel antibody and detection by immunoblot analysis. Cells were treated with 10 μM MG132 (proteasome inhibitor) for 6 h. (B) Flag-AMBRA1 and Myc-ALDH1B1 were cotransfected with HA-Ub into HCT116 cells and the cells were harvested after 6 h of treatment with 10 μM MG-132 or control (DMSO) treatment. Immunoprecipitation with an anti-c-Myc agarose affinity gel antibody was used to measure the ubiquitination of Myc-ALDH1B1 (n = 3). (C) The interaction of Myc-ALDH1B1 with Flag-DDB1 was identified by Co-IP using anti-Flag affinity gel (n = 3). (D) After overexpressing Myc-ALDH1B1 with Flag-TRAF6 or Flag-HUWE1 in HEK293T cells, the interaction was investigated by immunoprecipitation and immunoblot analysis using an anti-c-Myc agarose affinity gel antibody. The asterisk indicates Flag-TRAF6 in the immunoprecipitates (n = 3). (E) The ubiquitination of Myc-ALDH1B1 after overexpression of Flag-AMBRA1, Flag-DDB1, and Flag-TRAF6 was analyzed by a cell-based ubiquitination assay and MG132 treatment (10 μM, 6 h) (n = 3). The red asterisks indicate the overexpressed proteins.