Figure 5.

Knockdown of PHLPP2 promoted BECN1 protein degradation in UMUC3-MIR516Ai -Nonsense cells. (A) UMUC3-MIR516Ai-shPHLPP2#2, UMUC3-MIR516Ai -shPHLPP2#5, and UMUC3-MIR516Ai-Nonsense cells were cultured in 6-well plates until the cell density reached 70–80%. Following synchronization in 0.1% FBS DMEM for 12 h, the medium was replaced with 10% FBS DMEM for another 12 h. Then, total RNA was extracted using the TRIzol reagent, and the mRNA expression of BECN1 was detected by qPCR. (B and C) UMUC3-MIR516Ai-shPHLPP2#2, UMUC3-MIR516Ai-shPHLPP2#5, and UMUC3-MIR516Ai-Nonsense cells were subjected to determination of BECN1 protein degradation in the presence of the eukaryote protein synthesis inhibitor, CHX (50 μg/mL) by western blotting (B). The results are expressed as the mean ±SD from three independent experiments. An asterisk (*) indicates a significant decrease in comparison to UMUC3-MIR516Ai-Nonsense cells (p < 0.05) (C). (D) UMUC3-MIR516Ai-shPHLPP2#2 and UMUC3-MIR516Ai-Nonsense cells were pretreated with proteasome inhibitor MG-132 (10 μM) for 12 h, and then subjected to determination of BECN1 protein degradation in the presence of CHX (50 μg/mL) by western blotting. (E) UMUC3 cells expressing GFP-BECN1 or vector control GFP were co-immunoprecipitated with anti-GFP antibody and then subjected to MS analysis. Numbers of peptides identified by MS for BECN1 degradation-related proteins were listed as indicated. (F) Indicated UMUC3 cell transfectants were subjected to western blot for the determination of BECN1 degradation-related proteins. (G) UMUC3 cell transfectant lysates were subjected to Co-IP with an anti-GFP antibody. The binding proteins were determined by western blot. (H) UMUC3-MIR516Ai–Vector and UMUC3-MIR516Ai-Myc-CUL4A cells were subjected to the determination of BECN1 protein degradation in the presence of CHX (50 μg/ml) by western blot. (I) The knockdown of CUL4A in UMUC3-MIR516Ai-shPHLPP2#2 cells and BECN1 expression was assessed by western blot. (J) The BECN1 protein degradation was evaluated in UMUC3-MIR516Ai -shPHLPP2#2-Nonsense, UMUC3-MIR516Ai-shPHLPP2#2-shCUL4A#1 and UMUC3-MIR516Ai -shPHLPP2#2-shCUL4A#2 cells