Figure 5.

The PY motifs are essential for TMEPAI-mediated c-Maf ubiquitination. A, the PY motifs in TMEPAI. To make the PY motif mutation, Tyr was mutated into Ala as indicated. B, the wildtype and PY-mutated TMEPAI plasmids were co-transfected into HEK293T cells with c-Maf, respectively. Twenty-four hours later, the cells were treated with MG132, followed by immunoprecipitation and immunoblotting assay. C, the wildtype or PY-deleted TMEPAI plasmids were co-transfected into HEK293T cells with c-Maf, respectively. Twenty-four hours later, the cells were treated with MG132, followed by immunoprecipitation and immunoblotting assay. D, NEDD4 and TMEPAI plasmids were co-transfected into HEK293T cells; 48 h later, cell lysates were prepared for immunoprecipitation and immunoblotting assay. E, TMEPAI and its PY mutants were transfected into HEK293T cells; 48 h later, cell lysates were prepared for immunoprecipitation and immunoblotting assay. TCL, total cell lysates.