Figure 3

PRDX6 induces PrP upregulation. (A) Detection of endogenous PRDX6 in uninfected N2a58# and scrapie-infected N2a58/22L cells was performed by Western blot using specific anti-PRDX6 and anti-β-actin antibodies (upper panel). Quantification of PRDX6 expression was carried out of four independent experiments (lower panel; *, p < 0.05). (B) PrP (red) and PRDX6 (green) in uninfected N2a58# and scrapie-infected N2a58/22L cells were detected by laser scanning microscopy using PrP-specific 6H4 and PRDX6 antibodies. Nuclei (blue) were stained by DAPI. Scale bar, 10 μm (left panel); scale bar, 5 μm. (C) Downregulation of PRDX6 was performed by reverse transfection of a control siRNA (Ctr. siRNA) or a combination of two siRNAs specific for PRDX6 (PRDX6 siRNA). 48 h after transfection cells were lysed and either incubated with 20 μg/ml PK (+) or left untreated (−). Western blot was performed using specific antibodies against PRDX6, PRDX1-4, PrP (8H4) and anti-β-actin antibodies. (D) Cells were transfected with an empty vector control or a PRDX6 expression plasmid (PRDX6-flag) and lysed 24 h after transfection. Equal amounts of cell lysates were treated with PK (+) or left untreated (−) following SDS-PAGE and immunoblotting using anti-flag, anti-PRDX6, anti-PRDX1-4, anti-PrP (8H4) and anti-β-actin antibodies. (E) The activity of iPLA₂ was analyzed in N2a58# (black bars) and scrapie-infected N2a58/22L cells (grey bars), which were either treated with siRNA targeting PRDX6 or control siRNA (Ctrl) (*, p < 0.05; ***, p < 0.001)