Fig. 5
A29-V40 peptide activates Nox2 to produce superoxide via binding to gp91phox . a Membrane translocation of p47phox and p67phox in rat microglia-derived HAPI cells. After stimulation by PMA or α-Syn peptides A19-A30 and A29-V40, cells were lysed in hypotonic lysis buffer. The membrane fraction and the cytosol were extracted from the lysates and were dissolved in SDS-PAGE and transferred to PVDF membranes. Samples were immunoblotted for p47phox, p67phox, gp91phox, and tubulin. PM plasma membrane; Cyt cytosol. b Quantitative analyses of p47phox and p67phox membrane translocation according to the immunoblot data as shown in a. ANOVA was performed; n = 3–5; and *P < 0.05, **P < 0.01 compared to the corresponding basal controls. c Superoxide production in mouse microglial cultures. WT or gp91phox−/− microglia were seeded in 96-well plates. Upon stimulation using PMA (positive control) or α-Syn peptides A29-V40, superoxide production was measured. n = 4–6; *P < 0.05 and ***P < 0.001, compared to the corresponding basal control; ### P < 0.001, compared as indicated. d Representative images showing membrane translocation of WT mouse microglial p47phox. After stimulation using PMA or α-Syn peptides A19-A30 and A29-V40, the cells were fixed, permeabilized, and immunostained for CD11b and p47phox. e Representative images showing membrane translocation of gp91phox null microglial p47phox. After stimulation using PMA or α-Syn peptides A19-A30 and A29-V40, gp91phox null microglia were fixed, permeabilized, and immunostained for p47phox. f Binding of A29-V40 peptide to gp91phox. Flag-fused A29-V40 peptide was incubated with WT or gp91phox−/− microglial lysates, and the mixtures were further incubated with protein G-conjugated magnetic beads which were coated with control IgG or anti-gp91phox polyclonal Ab. After washes, beads were boiled and the supernatants were dissolved in SDS-PAGE. Samples were immunoblotted for Flag, gp91phox, or tubulin. g Binding of A29-V40 peptide or A19-A30 peptide to gp91phox. h Representative images illustrating the cell surface binding of FITC-labeled A29-V40 peptide but not A19-A30 peptide to gp91phox. WT or gp91phox−/− microglia were incubated with FITC-labeled A29-V40 peptide or A19-A30 peptide on ice for 30 min and imaged by a confocal microscope. Bar = 10 μm