Figure 3.

CUL3 promotes K48-linked ubiquitination of BECN1. (A) The ubiquitination of BECN1 decreased with CUL3 deletion. 293 t cells were transient transfected with siRNA targeting CUL3, incubated with 10 μM MG132 for 6 h, and then anti-BECN1 immunoprecipitation and immunoblotting was performed. (B) CUL3 increases BECN1 ubiquitination. 293 t cells were transfected with HA-ubiquitin, MYC-CUL3 and Flag-BECN1, and treated with 10 μM MG132 for 6 h. The immunoprecipitation and immunoblotting was performed by using the indicated antibodies. (C,D) The ubiquitination of BECN1 is K48-linked. 293 t cells were transfected with indicated plasmids, and immunoprecipitation with Flag affinity gels and immunoblotting were performed. (E) The prediction of BECN1 ubiquitination site by using Ubpred. (F) 293 t cells were transfected with HA-ubiquitin and different mutant BECN1 plasmids, and the immunoprecipitation with Flag affinity gels and immunoblotting were performed