Fig. 4: Rab11b recycling alters the surface proteome and controls integrin β1 localization and activation.

a Transferrin receptor recycling in MDA-231 cells. n = 3 independent experiments. Two-way ANOVA, Sidak’s multiple comparison. b Transferrin receptor recycling in MDA-231 cells co-cultured for two days. Cancer cells were selected on expression of CD-44. n = 3 independent experiments. Two-way ANOVA, Sidak’s multiple comparison. c Schematic of surface biotinylation. d Association of Rab11b with biotinylated surface proteins determined by immunoprecipitation and immunoblotting. e Retention of total or surface biotin following surface biotinylation with biotin-PE. n = 2 independent experiments. Two-way ANOVA, Sidak’s multiple comparison. f Schematic of biotinylated surface protein isolation and proteomics. g Correlation matrix of all measured samples based on Pearson’s correlation values. h Cleveland plot of top GO terms enriched in proteins that were decreased on the surface of shRab11b cells. i Heatmap of proteins annotated with GO term cell adhesion, containing at least one predicted transmembrane domain. j Representative images of H&E and IHC for mice intracranially injected with MDA-231-tdTomato cells. IHC for cytokeratin 8 (K8), Rab11b and integrin β1 (ITGB1). Scale bar 50 μm. k Immunoblotting of total and surface lysates. l Surface integrin β1 recycling in MDA-231 cells. n = 2 independent experiments. Two-way ANOVA, Sidak’s multiple comparison. m Linear regression of data in (p). Points, mean ± s.d., color shading, 95% confidence interval. Analysis of covariance. n–p MDA-231 cells stained for integrin β1 and actin (phalloidin, to delineate cell boundaries) (n), or active integrin β1 and actin (o). p Corrected total cellular active integrin β1 fluorescence (CTCF) was determined for individual cells. n = 3 independent experiments. Bars, mean ± s.d. Two-sided t-test. Scale bar 15 μm. For panels (a, b, e, and l) Boxes, first to third interquartile range, line, mean, points, outliers. For all panels, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.