Figure 3.

6-OHDA-induced oxidative stress was implicated in PARK7 secretion. (A) SH-SY5Y cells were treated with 100 μM 6-OHDA for 3 h and were subjected to GSH assay. n = 5; mean ± S.D.; **, p < 0.01. (B) SH-SY5Y cells were pre-treated with or without 2 mM NAC for 2 h and were then treated with 100 μM 6-OHDA for 3 h, followed by culture in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for PARK7 or RPN1. PARK7 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D.; *, p < 0.05. (C) SH-SY5Y cells were treated with 100 μM H₂O₂ in the presence or absence of 50 U/ml catalase in serum-free medium for 3 h. Whole cell lysates and the conditioned medium were immunoblotted with antibodies specific for PARK7, RPN1, or FN1. PARK7 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D. (D) SH-SY5Y cells were treated with 100 μM 6-OHDA for 3 h and were then cultured in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were separated by 2D-PAGE and immunoblotted using antibody specific for PARK7. The ratio of oxPARK7:total PARK7 is shown under each condition. (E) HEK293 cells stably expressing WT or C106S mutant of PARK7 were treated with 100 μM 6-OHDA for 3 h and were then cultured in serum-free medium for 2 h. Whole cell lysates and the conditioned medium were immunoblotted using antibodies specific for PARK7 or RPN1. PARK7 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; mean ± S.D.; *, p < 0.05.