Fig 2: CapZ facilitates the recruitment of RAB5 effectors to early endosomes.(A) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. (B, C) HEK293 cells were transiently transfected with Flag-Rabaptin-5 (B), Flag-VPS34 (C), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. (D) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. (E) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. (F) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. (G) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. (H) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. (I) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. (J) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1.Original immunoblots. Figure 5—source data 2.Data for 5D–5G, 5J.

Fig 3: AG-08 is related with endosomal pathway. (A) After pre-treatment with 20 µM pitstop II, 0.5 mM methyl-β-cyclodextrin (MβC), 80 µM dynasore, 20 µM cytochalasin d (Cyto D) or 20 µM chlorpromazine (CPZ) for 1 h, HCC1937 cells were treated with 8 µM AG-08 for 24 h. Then cell viability was determined by using WST-1. Reported values were normalized on cells treated with only AG-08. Error bars are the standard deviations (n = 3). p-values were calculated with respect to AG-08 treated cells by two-tailed equal variance Student’s t-test (*p < 0.05, ***p < 0.001, ****p < 0.0001) (B) HCC1937 cells were treated with brefeldin A (Bref A) for 1 h and then treated with 8 µM AG-08 for 24 h. Error bars represent standard deviations (n = 3). p-values were calculated with respect to AG-08-treated cells (*p < 0.05, **p < 0.005, ***p < 0.001). (C) Following AG-08 treatment for 16 h, EEA1 proteins were stained using the anti-EEA1 antibody in HCC1937 cells (scale bar = 25 µm).

Fig 4: SEC14L2 knockdown disturbs VEGFR2 internalization and recycling processes. a Western blotting results show impairment of biotinylated VEGFR2 internalization in SEC14L2 knockdown cells. VEGFR2-overexpressing HEK293T cells were transfected with NC or SEC14L2 shRNA for internalization assay described as supplementary Fig. 10. Quantified internalized VEGFR2 levels are shown on the right as mean ± SD from three independent experiments (n = 3). Red and blue lines represent control and SEC14L2 shRNA transfected group, respectively. TCL, total cell lysate. ***p < 0.001. b, c Analysis of VEGFR2 internalization based on immunostaining assay in HUVECs. HUVEC cells infected with NC or SEC14L2 shRNA were cultured for internalization assay and then cells were fixed for immunostaining with anti-EEA1 (white) and anti-SEC14L2 (green) antibodies. Quantification data of plasma membrane VEGFR2 (red) before internalization, internalized VEGFR2 after internalization, overlapping coefficient between internalized VEGFR2 and EEA1 (n = 40 cells) as well as EEA1 positive dots are shown individually in c. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. d Western blot analysis of VEGFR2 recycling over a 35 min time course. VEGFR2-overexpressing HEK293T cells were transfected with NC- or SEC14L2 shRNA for 48 h and used for recycling assay as detailed in Supplementary Fig. 10. The recycling rate of VEGFR2 was measured with or without 100 ng ml−1 VEGFa stimulation. Quantification data are shown as mean ± SD from three independent experiments on the right (n = 3). **p < 0.01; ***p < 0.001. e, f SEC14L2 knockdown promotes VEGFR2 localization in RAB7+ (e) or CD63+ (f) compartments. HUVEC cells infected with NC or SEC14L2 shRNA were transfected with RAB7-EGFP (e) or harvested directly (f) for anti-VEGFR2 (red) and EGFP/CD63 (green) staining. Regions in the boxes are enlarged in the right corner. Scale bars, 10 μm. The percentage of VEGFR2 co-localized with RAB7/CD63 is shown individually in the right, n indicates observed cell number. **p < 0.01; ***p < 0.001. Two-way ANOVA tests were used for a, d and t-tests for c, e, and f. Source data are provided as a Source Data file

Fig 5: Autophagy inhibition increases PI(3)P‐Positive early endosomesThe following experiments were performed in glial shNf‐1/shTp53 glial cells serum starved for 4 h before assaying. Control, sgAtg7 or sgAtg16l1 cells were treated with 2 ng/ml EGF for 15 min. Cells were then processed for staining using anti‐EEA1 antibodies and PI(3)P probe (Alexa 488‐labelled 2XFYVE domains). To ensure the specificity of the probe, control cells were pre‐treated with 5 mM 3'MA for 30 min. Scale bar: 10 μm.Quantification of PI(3)P+ label intensity per cell (in A).Pearson's colocalisation coefficient between PI(3)P and EEA1 (in A).Control and sgAtg7 cells were stimulated 20 ng/ml EGF for 5 or 15 min before immunofluorescence staining against EEA1 and EGFR. Scale bar: 10 μm.Pearson's colocalisation coefficient between EEA1 and EGFR (in D).Endogenous VPS34 was immunoprecipitated from control and sgAtg7 cells that were treated with 2 ng/ml EGF for 15 min and then lysed in CHAPS‐containing detergent buffer and binding partners detected by Western blotting.Densitometry analyses of proteins coimmunoprecipitated with endogenous VPS34 (in F).Data information: Statistical analyses were performed on at least three independent experiments, where error bars represent SEM and P values represent a two‐tailed Student's t‐test: NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.