WASH and exocyst complex associate on adjacent subdomains on MT1-MMP–positive endosomes. (A) Association of endogenous WASH and cortactin (Cttn) on microdomains on MT1-MMPmCh–positive late endosomes. (B) Zoom of boxed regions in A. Images represent the middle plane of the cells. (C) MDA-MB-231 cells transfected with GFP-WASH were plated on cross-linked gelatin, stained for Exo84 and MT1-MMP, and imaged by 3D deconvolution microscopy. (D) Zoom of boxed region in C (box 1) and from another cell (box 2). Bars: (A and C) 5 µm; (B and D) 1 µm. (E) Box plots of the distance between adjacent puncta of WASH and cortactin or Exo84 on MT1-MMP–positive endosomes (in pixels). Whiskers show 25–75%. Numbers of cells (n) and of WASH-cortactin and WASH-Exo84 doublets (d) analyzed from independent experiments are indicated. (F) Quantification of Duolink dots using different antibody pairs as indicated (mean Duolink dots per cell ± SEM; n represents the number of cells analyzed for each condition). ***, P < 0.001 as compared with WASH-Exo84 combination. (G) Immunolabeling of MT1-MMP (green) was performed after the Duolink reaction between WASH and Exo84 antibodies (red dots). Bar, 5 µm. (H) Coimmunoprecipitation of exocyst subunits with WASH in HeLa cell lysates. Bound proteins were analyzed by immunoblotting with antibodies against WASH, HA-tag, and Sec8. Input lysates (1%) were loaded as control.