Fig. 7: SHIP164 is required for Rab14-positive EE budding.

a Representative images of a fixed HEK293 cell stained with pre-cleared SHIP164 antibody (green), EEA1 antibody (blue), and phalloidin (magenta). An inset was shown on the right. b The distribution of endogenous SHIP164 puncta. 369 SHIP164 puncta from 16 cells. Data are represented as mean ± SD. c CLEM of a fixed HeLa cell transfected with Halo-Rab14 (magenta) and stained with phalloidin (green) and DAPI (blue) with an inset. Arrows indicate the base of RAB14-labeled endosomal bud marked by actin foci. d Schematic cartoon showing two EE structures, EE vacuole, and EE bud. e, f Representative images of fixed HEK293 cells probed with antibodies against endogenous Vps26B (magenta) and phalloidin (green) upon scrambled (e, left), SHIP164 siRNA (e, right) or Rab5 siRNA (f) with insets on the bottom. g, h The size (g) and number (h) of endosome buds are positive for both Vps26B and actin in cells. More than 20 cells were quantified for each condition from three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Data are represented as mean ± SD. i Immunoblots showing that SHIP164 depletion did not affect the level of Vps26B, or vice versa. j Representative images of fixed HEK293 cells probed with antibodies against endogenous EEA1 (magenta) and phalloidin (green) upon scrambled or SHIP164 siRNA with insets on the bottom. k Representative images of fixed HEK293 cells expressing Halo-Rab14 (magenta) and stained with antibodies against endogenous EEA1 (blue) and phalloidin (green) with insets and line-scan analyses on the right. l Representative images of two SHIP164 KO HEK293 clones expressing Halo-Rab14 with two insets on the right. Yellow arrows indicated Rab14 buds in control cells and red arrows denoted Rab14 enriched foci on EE in SHIP164 KO cells. m The percentage of Rab14 endosomes with buds in l. 1371 Rab14 EEs from 18 control cells, 478 Rab14 EEs from 18 SHIP164 KO #6, or 583 Rab14 EEs from 20 SHIP164 KO #9 cells were quantified from three independent experiments. Data are represented as mean ± SD. Scale bars: 10 μm in the whole cell images and 2 μm in the insets (a, e, f, j–l); 10 μm in the whole cell images and 1 μm in the insets (c).