Figure 2.

CUL3 attenuates proteasomal degradation of BECN1. (A) The protein lysates of the indicated cell lines were analyzed by western blot by using anti-BECN1 or CUL3. (B) The RNA of the indicated cell lines was extracted and the expression of BECN1 and CUL3 were analyzed by qRT-PCR. (C) CUL3 affects the protein expression of BECN1. MDA-MB-231 cells were transfected with siRNA targeting CUL3, or plasmids encoding CUL3, for 48 h. The protein extracts were subjected to the immunoblotting with indicated antibodies. (D) Knockdown of CUL3 does not affect the transcription of BECN1. MDA-MB-231 cells were transfected with siRNA targeting CUL3, and the expression of BECN1 or CUL3 was detected by qRT-PCR. (E) CUL3 regulates the degradation of BECN1 protein. Hela cells transfected with CUL3 siRNA were treated with 20 μM CHX for the indicated time, and the protein extracts were subjected to the immunoblotting assay. (F) CUL3 promotes the proteasomal degradation of BECN1. MDA-MB-231 cells transient overexpressing with MYC-CUL3 were treated with 100 nM BafA1 or 10 μM MG132 for 6 h, and then assayed for immunoblotting. CUL3 ubiquitin ligase inhibits autophagy. (G) MDA-MB-231 cells were transfected with siRNA targeting CUL3 for 48 h. Cells were treated with BafA1 for 6 h and the cell lysates were detected by immunoblot. (H) Hela cells overexpressing EYFP-LC3 were transfected with the indicated siRNAs for 48 h, and then be fixed and subjected to immunofluorescence assay. Scale bars: 10 μm. (I) MDA-MB-231 cells were transfected with the indicated siRNA for 48 h. Cell lysates were subjected to immunoblotting assay. (J) CUL3 does not affect the formation of BECN1 complex. 293 t cells transfected with siRNA targeting CUL3 and the immunoprecipitation was performed by using anti-BECN1 antibody. The formation of BECN1 complex was analyzed by western blot using the indicated antibodies