Characterization of endosomal association of ARP2/3 and WASH components, and the interaction of ARP2/3 with WDR91, and FAM21 with SNX27 or VPS35. (A and B) Co-immunostaining of endogenous ARPC2 with Rab7 in Ctrl (A) and KO-91 (B) cells treated without or with VPS34-IN1 (2 μM, 3 h). Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of linear pixel fluorescence along the arrows across the endosomes in the zoom images are shown on the right. Green: ARPC2; Magenta: Rab7. (C and D) Co-immunostaining of endogenous FAM21 with Rab7 in Ctrl (C) and KO-91 (D) cells treated without or with VPS34-IN1 (2 μM, 3 h). Zoom images show magnified frames in the merged images. Bars, 5 µm. Quantifications of linear pixel fluorescence along the arrows across the endosomes in the zoom images are shown on the right. Green: FAM21; Magenta: Rab7. (E and F) Co-IP of Flag-WDR91 with Myc-ARP2 (E) and Myc-ARP3 (F). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and Myc antibodies. (G and H) Co-IP of Flag-FAM21 with GFP-SNX27 (G) and GFP-VPS35 (H). IPs were performed with anti-Flag antibody resin and immunoblotted using Flag and GFP antibodies. Source data are available for this figure: SourceData FS5.