FIG. 6.
Nedd4 can be precipitated with N-terminal LMP2A GST fusion protein but does not associate with full-length LMP2A when both proteins are overexpressed in Cos-1 cells. (A) Wild type (YY) and PPPPY motif (AA, Y60A/Y101A; YA, Y101A; AY, Y60A) mutants of a FLAG epitope-tagged LMP2A mammalian expression vector were transfected into Cos-1 cells with the T7 epitope-tagged Nedd4 expression vector. Lysates were then subjected to anti-FLAG immunoprecipitations (IP), and associated protein was identified by SDS–8% PAGE with Western blotting (WB) for T7 epitope-tagged protein. (B) Cos-1 cell lysates containing T7 epitope-tagged Nedd4 protein were treated with immobilized GST or GST fusion proteins containing the N-terminal 122 amino acid residues of wild-type LMP2A or PPPPY mutants. Associated Nedd4 was identified by SDS–9% PAGE with Western blotting (WB) for the T7 epitope-tagged protein. WCL, whole-cell lysate.