Figure 3.

NEDD4 is required for TMEPAI-induced c-Maf polyubiquitination and degradation. A and B, a c-Maf plasmid was co-transfected with a TMEPAI plasmid into HEK293T cells along with a NEDD4 (A) or a WWP2 (B) plasmid for 24 h, followed by BZ treatment. Cell lysates were then subjected to the IP and IB assay. C, cell lysates from a panel of cancer cell lines were subjected to evaluation of the expression level of TEMPAI and NEDD4 by immunoblotting assay. D, HEK239T cells were transfected with NEDD4 for 24 h, followed by immunoblotting assay. E, HEK293T cells were infected with shNEDD4 for 48 h, followed by immunoblotting against specific proteins as indicated. F, HEK293T cells were transfected a NEDD4 plasmid for 24 h, followed by infection with shNEDD4. The cells were then incubated for another 48 h before being prepared for immunoprecipitation/immunoblotting assays as indicated. TCL, total cell lysates.