Figure 5

BEX2 is degraded by a proteasomal-dependent pathway. (A) BEX2-binding partners were determined by LC/MS analysis. Immunoprecipitation (IP) was performed using 293 T-FlpIn-T-REx-Flag-BEX2 cells. (B) Immunoprecipitation of FEM1B using anti-Flag antibody. Flag-BEX2 and myc-FEM1B were transfected into 293 T cells, and IP was performed. (C) HuCCT1 cells were stimulated with the lysosomal inhibitor (NH₄Cl) or proteasomal inhibitor (MG132) and the expression level of BEX2 was determined by western blot analysis. (D) The degradation rate of Flag-BEX2 in 293 T-FlpIn-T-REx-Flag-BEX2 cells was determined by western blot analysis. CHX, cycloheximide. (E) Immunoprecipitation assay of 293 T cells transfected with the indicated plasmids and immunoprecipitated with an anti-Flag antibody showed that Flag-BEX2 was ubiquitinated under the co-transfection of FEM1B and CUL2. (F) 293 T cells were transfected with the indicated plasmids and the expression level of Flag-BEX2 was determined by western blot analysis. (G) FEM1B expression was knocked-down by targeted siRNA in HuCCT1-BEX2 cells. Two days after transfection, cells were harvested and BEX2 expression was determined by western blotting.