Fig 2: SEC14L2 knockdown decreased VEGFR2 phosphorylation at Y1175 site. a Western blot analysis of cell lysates from NC or SEC14L2 shRNA infected HUVECs. Cells were infected and starved overnight, followed by 100 ng ml-1 VEGFa stimulation for 5, 15 or 30 min, respectively. VEGFR2 (p-Y1175), VEGFR2 (p-Y1054/1059), and total VEGFR2 levels were examined by immunoblotting using respective antibodies. b, c Statistical results of p-VEGFR2-Y1054/1059 (b) and p-VEGFR2-Y1175 (c) levels in a relative to total VEGFR2. Data are normalized to control group without stimulation and shown as mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. d, e Overexpression of zebrafish sec14l3 in HEK293T cells restores the level of p-VEGFR2-Y1175 inhibited by SEC14L2 knockdown. One or two micrograms sec14l3 plasmid was transfected into SEC14L2 knockdown stable HEK293T cells with VEGFR2 overexpression. Following starvation overnight, 100 ng ml-1 VEGFa was added and stimulation continues for 0, 5 or 15 min. Quantification data of the relative p-VEGFR2-Y1175 level from three repeated experiments are normalized to control group with 0’ stimulation and shown on the right (e). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. The loading volume of a, d was adjusted for the same amount of total VEGFR2 level in each lane. Two-way ANOVA tests were used for statistical analyses in b, c, and e. Source data are provided as a Source Data file

Fig 3: Sec14l3/SEC14L2 interact with VEGFR2 and RAB4A/5 A via complementary domains. a SEC14L2 interacts with VEGFR2 endogenously in HUVECs. HUVEC cells were starved and stimulated with 100 ng ml-1 VEGFa for 5 min before harvest for IP and immunoblotting. TCL, total cell lysate. IgG serves as a negative control. Quantification of the interaction from three independent experiments is shown on the right (n = 3). *p < 0.05. b, c Sec14l3 shows a much stronger association with WT-VEGFR2 than its kinase domain mutation form (KM-VEGFR2). Different forms of VEGFR2 were co-expressed with Flag-tagged Sec14l3 in HEK293T cells. WB, western blot. Quantification of the interaction is shown on the right (n = 3). t-tests were used for statistical analyses in a–c. d Schematic diagrams of Sec14l3 and its truncated mutants. Numbers above the diagram indicate the corresponding amino acid positions. ?N indicates N-terminal CARL-TRIO domain deletion; ?S, Sec14 domain deletion; ?G, GOLD domain deletion. e Sec14l3 interacts with VEGFR2 via its Sec14 domain. HEK293T cells were transfected with different Sec14l3 domain deletion forms with VEGFR2 plasmid respectively and harvested for IP. f Sec14l3 specifically interacts with RAB4A and RAB5A. Different RAB plasmids were co-transfected with VEGFR2 into HEK293T cells respectively for co-IP assay. g, h SEC14L2 interacts with RAB4 (g) and RAB5 (h) endogenously in HUVECs. i Sec14l3 interacts with RAB4A via its CRAL-TRIO and GOLD2 domains. Source data are provided as a Source Data file

Fig 4: Enhanced VEGFR2-Y1175 phosphorylation restores SEC14L2/sec14l3 depletion effects. a Sec14l3 specifically interacts with the cytoplasmic region of VEGFR2 in HEK293T cells. The cytoplasmic region of VEGFR1, VEGFR2 or VEGFR3 was individually transfected with Flag-tagged Sec14l3 into HEK293T cells for immunoprecipitation (IP) and immunoblotting. TCL, total cell lysate. b SEC14L2/Sec14l3 could alleviate the interaction between PTP1B and the VEGFR2-cyto in HEK293T cells. HA-tagged PTP1B and Myc-tagged VEGFR2-cyto were co-transfected into HEK293T cells with Flag-tagged Sec14l3 or SEC14L2 shRNA. After 72 hpf, cells were harvested and lysed for immunoprecipitation assay using an HA antibody. WB, western blot. c, d PTP1B knockdown rescues p-VEGFR2-Y1175 level inhibited by SEC14L2 knockdown in HEK293T cells. PTP1B shRNA1 or shRNA2 was transfected into SEC14L2 shRNA stable HEK293T cells with VEGFR2 overexpression. p-VEGFR2-Y1175 levels were checked after 100 ng ml-1 VEGFa stimulation for 0, 5 or 15 min. The loading volume was adjusted for the same amount of total VEGFR2 level in each lane. Quantification data of the relative p-VEGFR2-Y1175 level from three independent experiments are shown in d (n = 3). e The cytoplasmic region of VEGFR2 mRNA rescues luminal defects in sec14l3 morphants. Five nanograms std-MO or sec14l3-tMO2 in combination with 50 pg VEGFR2-cyto mRNA was injected into one-cell stage embryos from Tg(kdrl: GFP)s843Tg transgenic fish, and harvested at 30 hpf for the measurement of luminal diameters /ISV length. f Statistical result of the luminal diameters of DA, PCV, and ISV in e. 20 embryos of each group from one representative experiment were calculated and shown here. Three independent experiments were carried out. g, h ptp1b knockdown partially restores the lumen size of DA/PCV in zebrafish sec14l3 morphants. Tg(kdrl:GFP)s843Tg transgenic embryos were injected with 0.5 ng std-MO or sec14l3-sMO in combination with 5 ng ptp1b-tMO and harvested at 25 and 30 hpf for the measurement of luminal diameters. Twenty embryos of each group were calculated, and statistical data from three independent experiments are shown in h. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. ANOVA tests were used for statistical analyses in d, f,and h. Scale bars, 50 µm. Source data are provided as a Source Data file

Fig 5: 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