Fig 1: Mutations in the PX domain of SNX17 influencecargobinding.A, Western blot analysis of whole cell lysates and GFP co-immunoprecipitations (IP) of moxGFP and moxGFP-tagged SNX17 variants from HEK293T cells. Blots were probed with antibodies against GFP and VPS35L to determine VPS35L binding. B, quantification of co-immunoprecipitated VPS35L normalized to GFP levels and relative to moxGFP-SNX17 WT (mean ± s.d.; n = 3). Statistical analysis was carried out with one-way ANOVA + Dunnett multiple comparison test compared to cells transfected with moxGFP-SNX17 WT. C, Western blot of a streptavidin-bead pull-down assay with biotinylated β1-integrin cytoplasmic tail peptides (Itgb1-peptide) or scrambled peptides and cell lysates from moxGFP-SNX17 WT, S38A or S38D cells. After pull-down, bound proteins were detected by Western blotting with antibodies against SNX17 and Kindlin-2. D, quantification of moxGFP-SNX17 binding to biotinylated β1-integrin cytoplasmic tail peptides normalized to bound Kindlin-2 (mean ± s.d.; n = 3). Statistical analysis was carried out with one-way ANOVA + Dunnett multiple comparison test compared to moxGFP-SNX17 WT. E, schematic domain organization of SNX17, SNX27, and the chimeric protein SNX17 PXSNX27, in which the PX domain of SNX17 was replaced by the PX of human SNX27. F, Western blot analysis of SNX17 KO fibroblasts re-expressing indicated SNX17 PX variants. Actin served as loading control. G and H, quantification of surface levels of Itgb1 (G) and Itga5 (H) on SNX17 KO fibroblasts re-expressing indicated SNX17 PX variants analyzed by flow cytometry (mean ± s.d.; n = 3). Statistical analysis was carried out with one-way ANOVA + Dunnett multiple comparison test compared to cells expressing moxGFP-SNX17 WT. I, Western blot of streptavidin-bead pull-down assay of biotinylated β1-integrin cytoplasmic tail peptides or scrambled peptides and lysates of SNX17 KO HeLa cells stably expressing moxGFP-SNX17 WT or moxGFP-SNX17 PXSNX27. After pull-down, bound proteins were detected by Western blotting with antibodies against GFP (to identify moxGFP-SNX17) and Kindlin-2. J, quantification of moxGFP-SNX17 binding to biotinylated β1-integrin cytoplasmic tail peptides (mean ± s.d.; n = 3). Statistical analysis was carried out with unpaired t test (two-tailed) with 95% confidence interval.