FIGURE

Fig. 3

ID

ZDB-FIG-251023-41

Publication

Kim et al., 2025 - APOA1 binding protein promotes lymphatic cell fate and lymphangiogenesis by relieving caveolae-mediated inhibition of VEGFR3 signaling

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Fig. 3

Cholesterol regulates VEGFR3 signaling.

a−d Effect of MβCD on VEGFR3 phosphorylation. a hLECs were growth factor-starved, and treated with 10 mM MβCD for 5, 15, and 30 min, and the resulting cells were further stimulated with 100 ng/mL VEGFC. The resulting cells were lysed and blotted using CAV-1, VEGFR3, GAPDH antibodies. b Quantitative analysis of panel a. Mean ± SD, n = 3 repeats; two-way ANOVA with Dunnett’s post-hoc test. c, hLECs were treated as in panel a. and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. d Quantitative analysis of (c). n = 3 repeats. Data are presented as mean ± SD and were analyzed using one-way ANOVA with Tukey’s post-hoc test. e, f Effect of AIBP treatment on VEGFR3 distribution in caveolar fractions. e hLECs were incubated with either recombinant AIBP or vehicle control in EBM2 supplemented with 10% FBS for 2 h, and the cells were subjected to sucrose gradient ultracentrifugation. n = 3 repeats. The resulting fractions were collected for Western blot analysis as indicated. Tx treatment; cav: caveolar fraction; n.c non-caveolar fraction. f Quantitative data of (e). Mean ± SD; two-way ANOVA with Sidak’s post-hoc test. n = 3 repeats. g, h Effect of AIBP and HDL co-treatment on VEGFR3 signaling. g hLECs were growth factor-starved and treated with HDL, AIBP, or HDL and AIBP in combination, and further stimulated with VEGFC. The resulting cells were lysed and immunoblotted as indicated. h Quantitative data of ERK and AKT activation. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 repeats. Ctrl: control. i Maxi-projection confocal images of Prox1⁺ and pErk1/2⁺ cells in the apoa1bp^−/−; fli1a:egfp zebrafish at 36 hpf following immunostaining using GFP, pErk1/2, and Prox1 antibodies. Dorsal (DA) aorta and cardinal vein (CV) were imaged. Arrows show the Prox1⁺ LECs with pErk1/2 expression. j Quantitative data of pErk1/2 intensity in Prox1⁺ LECs. Data are Mean ± SE; unpaired two-sided t-test with Welch’s correction. n = 146 (control) and n = 166 (apoa1bp^−/−) cells. Scale bar: 50 µm. Source data are provided as a Source Data file.

Expression Data

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Antibody Labeling

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Acknowledgments

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