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: In phKCs, KD of IL-13 receptors results in increased transcription levels of IL-20 and reduced levels of IL-20 receptors. Injection of IL-20 and IL-13 in mouse cheek induces itch-like behavior, though only when co-injected. (A) Lentiviral shRNA-mediated KD of IL-13Ra1 and IL-13Ra2 in phKCs. SNAP-23 serves as the internal control in Western-blotting. Percentage (%) KD relative to scrambled controls from n = 3 independent experiments. (B) Transcriptome analysis of IL-20 and its receptors in phKCs after KD of IL-13 receptors. KD cells and scrambled control-treated control cells were incubated with vehicle (left panel) or IL-13 (right panel) before RNA-seq. (C) Scratching bouts in mouse cheek model after injection of vehicle, or IL-13, or IL-20, or following co-injection of IL-13 and IL-20 (2-way ANOVA followed by Bonferroni's post hoc analysis; data are presented as mean ± SEM; n = 8 mice/group.). For (A) and (B) ns p > .05, *p < .05, **p < .01, and ***p < .001, a two-tailed Student's t-test followed by Welch's correction was used

Fig 4: 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 5: Representative images of SNAP23 distribution in human skeletal type I and nontype I muscle fibers. SNAP23 distribution is shown in cross sections (A) with the slow twitch fiber-type stain anti-MHC I (B). Images were obtained using a 40× objective of a widefield microscope. Bar = 50 µm. There was no difference in the fluorescence intensity of SNAP23 between type I and nontype I (C) where type I fibers were positively labeled with anti-MHCI. Values in panel C are given as means ± SEM.