Fig. 2. SNX27-retromer cargo is mis-sorted into lysosomes and degraded in the absence of SNX27 or VPS35.

(a) Immunofluorescent analysis of CD97 and TrailR1 in SNX27 and VPS35 depleted HeLa cells (green) with lysosomes stained with a LAMP1 antibody (red). (b) SNX27 and VPS35 suppressed cells were incubated with the lysosomal protease inhibitor Leupeptin (100μM) and antibodies against the ectodomain of TrailR1 and CD97 for 6h, followed by fixing, permeabilization and staining of lysosomes (LAMP1) and the internalized antibodies. Colocalization of internalized antibody and LAMP1 was quantified, the graph represents the mean of three independent experiments with 10 images each (n=3,* indicates p<0.05, unpaired t-test; error bars = s.d., scale bar = 10μm, individual data points shown in statistics source data) (c) Immunofluorescent staining and colocalization analysis of endogenous Glut1 glucose transporter and lysosomal marker LAMP1 in SNX27 and VPS35 deficient Hela cells. The graph represents the mean of three independent experiments with 10 images each. (n=3, * indicates p<0.05, unpaired t-test; error bars = s.d., scale bar = 10μm, individual data points shown in statistics source data) (d) Degradation assays of biotin labeled SNX27-retromer cargo. HeLa cells were transfected with siRNA against SNX27 and VPS35 and surface biotinylated 24h post transfection, before the effects of suppression became fully evident. Biotinylated proteins were captured from lysates with streptavidin beads at indicated timepoints after biotinylation and subjected to quantitative western blotting on an odyssey scanner. The plots represent the mean of 3 (ATP7A, GLUT1, TfnR, MCT1) or 4 (N-Cadherin) independent experiments. (* indicates p<0.05, unpaired t-test; error bars = s.e.m., individual data points shown in statistics source data, uncropped blots in Fig S5)