FIGURE 7.

Expression of catalytically inactive AMSH or UBPY mutants enhances PAR₂ ubiquitination under basal conditions. HEK cells were transiently transfected with PAR₂ and pcDNA5 (control), GFP-AMSH, GFP-AMSH(D348A), GFP-UBPY, or GFP-UBPY(C786S). Cells were unstimulated. PAR₂ was immunoprecipitated (IP), and membranes were analyzed by Western blotting (WB) for ubiquitin (ubi) and PAR₂. There was a low base-line level of PAR₂ ubiquitination in cells expressing empty vector (pcDNA5) that was unaffected by overexpression of AMSH or UBPY. Overexpression of AMSH(D348A) or UBPY(C786S) markedly increased basal levels of PAR₂ ubiquitination. Shown is a representative blot of n = 4 experiments.