Analysis of Y mutation in the tail of Itgβ1 and effects of SNX17 depletion on cell migration. (A) Immunofluorescent staining and quantitative colocalization analysis of endogenous LAMP1 and lentivirally expressed Itgβ1 with the indicated mutations in Itgβ1−/− MEFs. Error bars represent the standard deviation of twelve images from two experiments. (B) Degradation assay of the Itgβ1 proteins under cycloheximide (Cyclohex.)-treated conditions. Error bars represent the SEM of three experiments. (C) Western blot showing the effect of 14-h bafilomycin treatment on a mature form of mutated Itgβ1. (D) Integrin and SNX17 levels in control and SNX17-depleted fibroblasts. (E) Vinculin-stained fibroblasts spread on fibronectin for 10–90 min and analyzed for total focal adhesion (FA) area per cell (dotted line shows SNX17 knockdown; continuous line shows control cells) and the mean size of individual focal adhesions. (F) Migration tracks and mean speed and persistence values of fibroblasts migrating through a cell-derived matrix. Persistence shows linear displacement/total distance moved. Error bars represent SEM of >60 cells per condition. Bars, 20 µm. *, P < 10−5. Blots are representative of three experiments. Ctrl, control; IB, immunoblot; KD, knockdown; WT, wild type.
