Figure 3
Lack of arginylation leads to α-syn accumulation in cultured cells independently of proteasome degradation and renders insensitivity to inhibitors of autophagy. (A) Western blot quantification of the levels of unmodified α-syn (U) and α-syn mutants E46A, E83A, and E46AE83A transfected into cultured cells and analyzed in whole cell extracts (top) and pellets (bottom). The results for quantification were normalized by qPCR and tubulin loading control. (B) Western blot quantification of intracellular levels of α-syn transfected into wild type and Ate1 knockout cultured mouse embryonic fibroblasts. (C) Western blot quantification of intracellular levels of α-syn transfected into wild type and Ate1 knockout cultured mouse embryonic fibroblasts, analyzed in the absence or presence of proteasome inhibitor MG132 (6-hour treatment). (D,E) Western blot quantification of intracellular levels of α-syn transfected into Ate1 knockout cultured mouse embryonic fibroblasts, analyzed in the absence or presence of chloroquine (D) or bafilomycin (E). Bottom rows in all panels show representative immunoblots with α-syn and the loading control. For all panels, error bars represent SEM from 2 (panel C) and 3 (panels B, D and E) independent experiments, analyzed in duplicates.
