SNX17 binds to the membrane-distal NPXY motifs in β integrins. (A) Confocal image of internalized antibody (TS2/16) against active Itgβ1, endogenous EEA1, and GFP-tagged SNX17 in human RPE1 cells after 30 min of internalization. The boxed area is magnified below the merged image. Bar, 20 µm. (B) Western blot analysis of SNX17 in an immunoprecipitation of endogenous Itgβ1. (C) GFP-Trap precipitation of retrovirally expressed full-length Itgβ1-GFP from Itgβ1^−/− MEFs and Western blot analysis of endogenous SNX17 in the precipitates. The GFP blot shows expression of Itgβ1-GFP and GFP. (D) GFP-Trap precipitations of transiently transfected GFP-tagged cytoplasmic tails of indicated integrins from HEK293 cells. The blot shows endogenous SNX17 that precipitated with the Itgβ1 cytoplasmic tail. (E) Blot showing endogenous SNX17 in GFP-Trap precipitations of GFP-tagged integrin cytoplasmic tails. (F) GFP-Trap precipitations of Y motif mutated Itgβ1 and β5 cytoplasmic tails. (G) GFP-Trap precipitation of in vitro translated Itgβ1 cytoplasmic tails with Y motif mutations and the in vitro translated mCherry-tagged SNX17-FERM domain. (H) RFP-Trap precipitations of mCherry-tagged SNX17 domains and GFP-tagged Itgβ1 cytoplasmic tails with Y motif mutations. (I) GFP-Trap precipitation of SNX17 and SNX27 and Western blot analysis of WASH components. (J) Analysis of Itgα5β1 levels in a HeLa cell with suppressed expression of indicated sorting nexins and retromer components. IP, immunoprecipitation; IB, immunoblot; Strump., Strumpellin.