Figure 1.

PARK7 was secreted from SH-SY5Y cells. (A and B) SH-SY5Y cells were cultured in serum-free medium for 0–6 h. (A) Whole cell lysates (Cells) and the conditioned medium (Medium) were immunoblotted using antibodies specific for PARK7, RPN1, or FN1. Representative image is shown. (B) PARK7 band intensities were quantified by densitometric scanning and the percentage of secreted PARK7/total PARK7 is shown. LDH release in the conditioned medium was analyzed by LDH assay. n = 3; mean ± S.D.; *, p < 0.05; **, p < 0.01. (C) SH-SY5Y cells were treated with 2 μg/ml brefeldin A in serum-free medium for 3 h. Whole cell lysates and the conditioned medium were immunoblotted with antibodies specific for PARK7 or FN1. PARK7 and FN1 band intensities were quantified by densitometric scanning and relative secretion level to vehicle-treated cells is shown. n = 3; **, p < 0.01; n.s., not significant. (D) SH-SY5Y cells were homogenized by using the Dounce homogenizer and homogenate was sequentially centrifuged as indicated. Equal aliquots from each fraction were immunoblotted using antibodies specific for PARK7, LMNA (lamin A/C), VDAC1, RPN1, or proCASP3 (caspase 3). (E) SH-SY5Y cells were cultured in serum-free medium for 3 h. Whole cell lysates and the conditioned medium were separated by 2D-PAGE and immunoblotted using antibody specific for PARK7. The ratio of oxPARK7 to total PARK7 is shown under each condition.