Fig. 5: Vps26B is a Rab14 effector at EE buds. | Cell Discovery

Fig. 5: Vps26B is a Rab14 effector at EE buds.

From: Biogenesis of Rab14-positive endosome buds at Golgi–endosome contacts by the RhoBTB3–SHIP164–Vps26B complex

Fig. 5

a Representative images of fixed HEK293 cells stained with Vps26B antibody (green) and expressing Halo-Rab14 (magenta) with one large inset on the bottom and two small insets on the right. b Pearson’s correlation coefficient of endogenous Vps26B vs endosomal markers: Lamp1 (20 cells), GFP-FYVEX2 (13 cells), Rab10 (12 cells), Rab4A (12 cells), Rab5A (14 cells), Rab14 (32 cells), or FYVEx2 (13 cells) in 3 independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Data are represented as mean ± SD. c Representative images of fixed HEK293 cells stained with Vps26B antibody (green) and expressing Halo-Rab14 (blue) along with SNAP-VAPA (magenta). An inset was shown on the bottom with line-scan analyses on the right. d The distribution of endogenous Vps26B puncta relative to ER-Rab14 EE junctions. (1019 Vps26B puncta from 18 cells). Data are represented as mean ± SD. e, f Representative images of fixed HEK293 cells stained with Vps26B antibody (green) and expressing either Halo-Rab14 Q70L (magenta, e) or Halo-Rab14 N124I (magenta, f) with an inset on the bottom. g Pearson’s correlation coefficient of endogenous Vps26B vs either Rab14 N124I (26 cells) or Rab14 Q70L (14 cells), in more than three independent experiments. Two-tailed unpaired Student’s t-test. Data are represented as mean ± SD. h GFP-Trap assays demonstrate interactions between Halo-Rab14 mutants and endogenous Vps26B in HEK293 cells. i GST-pull-down assays show that GTP-loaded Rab14, but not Rab14-GDP, Rab4-GTP, or GST tag alone, binds to His-Vps26B. The Coomassie blue staining of proteins used in the assay is shown at the bottom. j GST-pull-down assays demonstrate that purified Rab14 and Rab14 Q70L, but not GST tag, or dominant-negative mutants S25N or N124I, were pelleted with purified His-Vps26B in vitro, with Rab4 as a negative control and SHIP164-890-CT as a positive control. The Coomassie blue staining of proteins used in the assay is shown at the bottom. Scale bars: 10 μm in the whole cell images and 2 μm in the insets (a, c, e, f).

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