Supplementary Figure 6

(a) CapZ mutant has impaired binding activity to PI3P. Co-sedimentation assay with PI3P micelles and recombinant WT CapZαCapZβ and CapZα(K256A, R260A)CapZβ(R225A). Proteins in supernatant (S) and precipitate (P) were visualized by SDS-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. The experiment was repeated 3 times. Uncropped images of gels are shown in Supplementary Fig. 9. (b) GFP-DFCP1-expressing NRK cells were transfected with wild type or mutant CapZα(K256A, R260A)-Myc and CapZβ(R225A)-Myc. Cells were then starved for 2 h and stained with antibodies to GFP and Myc. Regions outlined with white dashed lines are magnified. Scale bars in full panels and zoomed panels correspond to 5 μm and 1 μm, respectively. (c) Cells from (b) were quantified for CapZ-positive DFCP1 puncta. (n = 3 independent experiments; 50 cells were assessed per independent experiment.) Data represent mean ± s.d. ^∗∗P < 0.01 (two-tailed t-test).