Fig 2: Model of phagosomal turnover of gp91phox. (1) During phagosome formation, cytochrome b558 [gp91phox (gp91) and p22phox (P22)] is internalized from the plasma membrane together with zymosan. (2) Cytochrome b558-positive phagosomes contain the 3-phosphoinositides required for NOX2 activity – PI(3,4)P2 and/or PI(3)P. (3) NOX2 produces ROS, which result in auto-oxidation of cytochrome b558. (4) Phagosomal cytochrome b558 is replenished from an intracellular pool residing in LAMP1-rich late-endosomes/lysosomes through the action of the SNARE proteins VAMP8, stx7 and SNAP23. p47, p47 phox; p67, p67 phox; p40, p40 phox.

Fig 3: Gp91phox colocalizes with SNAREs on the phagosomal membrane. (A) Confocal images of dendritic cells stimulated with zymosan particles for 1 h and immunostained for gp91phox (green in merge) together with Vti1b, stx7, VAMP8, stx8, SNAP23, stx12 or stx5 (magenta). Yellow arrowheads, phagosomes positive for both gp91phox and the indicated SNARE. BF, brightfield. Scale bar: 5 µm. (B) Pearson correlation coefficients for colocalization of gp91phox with the indicated SNAREs for zymosan-containing phagosomes (from panel A) and for unstimulated cells (w/o zymosan; Fig S2A). Colocalization of gp91phox immunostained with both Alexa-Fluor-488 and -568-labeled secondary antibodies is shown as a positive control (individual donors shown). (C) Significance levels (one-way ANOVA with Tukey's test) from panel B. Black text, zymosan-pulsed samples; red text, unstimulated samples. ns, not significant. (D) Normalized staining intensity values of phagosomal gp91phox as a function of normalized intensity values of the indicated SNAREs for individual phagosomes (log2 scale; phagosomes pooled from >3 donors are shown; solid lines, linear regression with 95% confidence intervals; ß, regression coefficients; R2, R-squared values). (E) Quantification of the phagosomal enrichment for gp91phox and/or the indicated SNAREs (from panel D, with normalized intensity values >1). (F) Phagosomal enrichment of gp91phox and the indicated SNAREs following stimulation with zymosan (n=6 donors for gp91phox; n=3 donors for the SNAREs). Results show means±s.e.m.

Fig 4: Complex formation of stx7, SNAP23 and VAMP8. (A) Immunoprecipitation (IP) of stx7 (a-stx7) from zymosan-pulsed dendritic cells with or without NEM and probed for SNAP23 (a-SNAP23). Shown are a representative western blot and the quantification of the band intensities normalized to control conditions (without zymosan and NEM) (mean±s.e.m. of five donors; one-way ANOVA with Tukey's test). WCL, whole-cell lysate. First lane shows only antibody in buffer without lysate. Results are mean±s.e.m. (B) In vitro complex formation of purified full-length VAMP8 (blue arrowhead), SNAP23 (green) and stx7 (red) analyzed by SDS-PAGE and Coomassie staining. Ternary SNARE complexes (multiple bands between ~250 and 37 kDa; pink arrowheads) are SDS resistant at 20°C but disassemble at 95°C. A representative gel from three experiments is shown.