Figure 7
UBQLN2 targets pS129 α-syn to the proteasome for degradation. (A) Representative western blot of TKO cells overexpressing α-syn and transfected with either EV or UBQLN2 and treated with 10 μM lactacystin (Lac), 50 μM rolipram (Rol), or vehicle (Veh). Quantification of total α-syn and pS129 levels shows that pS129 accumulates in cells transfected with UBQLN2 and treated with Lac while total α-syn levels were not changed between groups (n = 9–10). Total α-syn and pS129 levels were each normalized to α-syn levels of EV, vehicle-treated cells. Western blots were cropped to focus on protein(s) of interest. (B) Representative images showing pS129 fluorescence in cells transfected with either EV or UBQLN2 and treated with either Veh, Lac, or Rol. Quantification of pS129 fluorescence intensity further supports that pS129 α-syn is increased when treated with Lac and in the presence of UBQLN2 (n = 11). pS129 fluorescence intensity was normalized to that of vehicle treated, EV cells. Scale bar 50 μm. (C) Representative western blot of TKO cells overexpressing α-syn and transfected with either EV or UBQLN2 and treated with 10 μM chloroquine (CLQ), 300 nM rapamycin (RAP), or vehicle (Veh). Quantification of pS129 or total α-syn levels reveals no differences between treatment groups (n = 10). Total α-syn and pS129 levels were each normalized to α-syn levels of EV-transfected, vehicle-treated cells. Uncropped Western blots are shown in Supplemental Figure S5.
