Additional Supporting Information may be found in the online version of this article.

CM_21069_sm_SuppFig1.pdf470KSupporting Information Figure 1. WASH silencing in NIH3T3 and MEF cells. Immunoblots loaded with equal amounts of total protein from either NIH3T3 (left) or MEF cells (right) that were treated with non-specific siRNA (siNS) or one of two siRNAs targeting WASH (siWASH 1, 2). Proteins were separated by SDS-PAGE and immunoblotted with antibodies against WASH and tubulin. Cells treated with either WASH siRNA exhibited an 80% reduction in WASH protein levels.
CM_21069_sm_SuppFig2.pdf470KSupporting Information Figure 2. FRAP analysis of α5-integrin-GFP and α-actinin-GFP dynamics. Cells were treated with two independent siRNAs targeting WASH (siWASH1, 2) or with control nonspecific siRNA (siNS). Following photobleaching, fluorescence recovery curves were obtained for: (A) focal adhesion associated α5-integrin-GFP, (B) non-focal-adhesion associated α5-integrin-GFP, and (C) focal adhesion localized α-actinin-GFP. (D) Quantification of percent recovery and half time to recovery (t½) for each condition.
CM_21069_sm_SuppFig3.pdf326KSupporting Information Figure 3. WASH silencing does not affect lamellipodia dynamics. Graphs depicting (A) the number of lamellipodia per cell, (B) lamellipodia protrusion velocity, (C) lamellipodia retraction rate, and (D) lamellipodia protrusion persistence time for NIH3T3 cells treated with non-specific siRNAs (siNS) or siRNAs targeting WASH (siWASH 1, 2). The graphs show the mean values and the error bars represent s.d. A student's t-test was performed and no significant difference was observed for any of the measured parameters between cells treated with siNS, siWASH 1 or siWASH 2.
CM_21069_sm_SuppMovie1.mov468KSupporting Information Movie 1. WASH and F-actin dynamics on subdomains of Rab5-Q79L endosomes. NIH3T3 cells transfected with pGFP-WASH (green), pLifeact-BFP (blue) to mark F-actin, and pDsRed-Rab5-Q79L (red) were imaged on a spinning disc confocal microscope.
CM_21069_sm_SuppMovie2.mov102KSupporting Information Movie 2. WASH and F-actin dynamics on subdomains of an enlarged endosome. Magnified from Movie 1, corresponding to the inset of Figure 1A.

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