Chimeric HECT ligases catalyze varying levels of Lck-Gag-PY-3K ubiquitination and autoubiquitination.

<p>Quantitative Western blot analysis of lysine-containing Lck-Gag-PY-3K proteins (A) and chimeric HECT ligase proteins (B), immunoprecipitated from 293T cells following cotransfection with plasmids expressing Lck-Gag-PY-3K, HA-ubiquitin, and the indicated chimeric YFP-WWP1/HECT ubiquitin ligases. The αPFV Gag immunoprecipitates were probed with an αHA monoclonal antibody (A, top panel) or αPFV serum (A, middle panel). The αGFP immunoprecipitates were probed with αHA (B, top panel) and αGFP (B, middle panel) monoclonal antibodies. Alternatively, unfractionated cell lysates were probed with αPFV serum or αGFP antibody (A and B, bottom panels). Lanes marked “none” contain immunoprecipitates or lysates from cells transfected with unfused YFP in place of a YFP-fused ubiquitin ligase. Note that the unfused YFP is not visible in these lanes because is migrates to a different position on the blot. Charts below each plot show quantitation (mean+SD of two experiments) of the total signals in each lane of the blots in the upper panels that were probed for HA-ubiquitin. The levels of Gag ubiquitination and autoubiquitination are presented relative to that observed in the presence of intact WWP1, which was assigned a value of 1.