FIGURE 8.

AMSH or UBPY knockdown prevent lysosomal trafficking, degradation, and deubiquitination of PAR₂. HEK-PAR₂ cells were treated with control (CON), AMSH, or UBPY siRNA and used for experiments after 72 h. A, Western blots show efficient knockdown of endogenous AMSH or UBPY. The lower panels show densitometric analyses of AMSH, UBPY, and β-actin. n = 3. *, p < 0.05. B, cell surface PAR₂ was labeled with FLAG antibody. Cells were challenged with AP (100 μm, 120 min), and PAR₂, LAMP1, and EEA1 were localized. In control siRNA-treated cells, PAR₂ colocalized with LAMP1. UBPY siRNA treatment diminished PAR₂ colocalization with LAMP1 and caused retention of PAR₂ in enlarged endosomes containing EEA1. Scale bars, 10 μm. C, cell surface proteins were biotinylated, cells were stimulated with AP (0 or 5 h), and biotinylated proteins were precipitated using avidin beads. PAR₂ and TfR were determined by Western blotting. PAR₂ degradation was significantly reduced in cells treated with AMSH or UBPY siRNA compared with control siRNA. n = 3. *, p < 0.05. D, cells were stimulated with AP (100 μm; 30 or 150 min), PAR₂ was immunoprecipitated (IP) and membranes were analyzed by Western blotting (WB) for ubiquitin (ubi) and PAR₂. In cells treated with control siRNA, PAR₂ was ubiquitinated at 30 min and deubiquitinated at 150 min. In cells treated with AMSH or UBPY-siRNA, PAR₂ remained highly ubiquitinated at 150 min. n = 3. *, p < 0.05. IP, immunoprecipitation; WB, Western blot.