Supplementary Figure 4: SCRIB required for clearing S. aureus infection in vivo and is recruited to phagosomes to mediate production of ROS.

(a) Immunoblot analysis for SCRIB and GFP expression in whole blood from WT, ishSCRIB mice. (b) Whole blood from WT, ishSCRIB + Dox or − Dox mice was incubated with S. aureus for 20 min. Lysotaphin was added at 20 min of incubation and aliquots were removed at 60 min for enumeration of S. aureus CFU. Blood from n = 3 independent mice were used and error bar represents mean ± SD. (two-tailed, unpaired t-test with equal variances: P = 0.001. (c) Cells were imaged every 40 s for 2.0 h. FITC containing phagosomes were detected at f + 80 s (where f is frame 116 out of 271, an arbitrary frame chosen that precedes observation of phagocytic events after addition of S. aureus). Concomitant accumulation of RFP-SCRIB around the FITC-S. aureus(t = 480 s) intensity that localized to and around the site of bacterial engulfment as monitored with the FITC S. aureus signal, white arrow. Scale bar is 5 μm. d Quantification of FITC S. aureus (green line) and RFP-SCRIB (red line) signal intensity at the point of phagocytosis that correlates with the time frames shown in c. Increases in RFP-SCRIB intensity largely follow the same pattern and colocalize with FITC S. aureus at the time of phagocytosis. e Cells were incubated with phrodo-S. aureus for 1 h and then fixed for immunofluorescence staining. Representative and magnified inset images are shown. Green is pHrodo-S. aureus; red is SCRIB. Scale bar is 5μm. f RAW 264.7shSCRIB cells lacking endogenous SCRIB were used to re-express wild type SCRIB (WT.Rescue), or with SCRIB with point mutation in the LRR domain (P305L) (P305L.Rescue). n = 3 biologically independent experiments (mean ± SD). (two-tailed, unpaired t-test with equal variances: P = 0.01). Unprocessed scans of full blots/gels are shown in Supplementary Fig. 6.