Journal: bioRxiv

Article Title: AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

doi: 10.1101/2020.10.16.342998

Figure Lengend Snippet: ( A and B ). Cholesterol removal potentiates VEGFR3 signaling. ( A ) hLECs were serum-starved, and treated with 10 mM MβCD for 30 min, and cells were further stimulated with 100 ng/ml VEGFC. The resulting cells were lysed and blotted with Cav-1 or GAPDH antibodies. ( B ) hLECs were treated as in ( A ). and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. ( C ) AIBP-mediated cholesterol efflux disrupts caveolae and reduces CAV-1 levels in the caveolar fractions. hLECs were treated with recombinant 200 ng/ml AIBP, 100 μg/ml HDL 3 , or both in serum-free EBM2 for 6 hours, and the cells were subjected to sucrose-mediated ultracentrifugation. The resulting fractions were collected for Western blot analysis as indicated. ( D ) AIBP-mediated cholesterol efflux increases VEGFR3 signaling. hLECs were serum-starved and treated as in ( C ), and further stimulated with 100 ng/ml VEGFC. The resulting cells were lyzed and immunoblotted as indicated. ( E and F ), Quantitative data of AKT activation ( E ) and ERK activation ( F ). Mean±SD, n=3 independent repeats. *, p<0.05; **, p<0.01; ns: not significant.

Article Snippet: Human dermal lymphatic microvascular endothelial cells (hLECs) (Angio-Proteomie, Cat # cAP-0003) were cultured in Endothelial Cell Medium (ScienCell, Cat # 1001).

Techniques: Immunoprecipitation, Western Blot, Recombinant, Activation Assay

Journal: bioRxiv

Article Title: AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

doi: 10.1101/2020.10.16.342998

Figure Lengend Snippet: ( A ) Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). ( B ) VEGFR3 AAA loses its binding to CAV-1. hLECs were transfected with control EGFP (Ctrl), VEGFR3-EGFP (R3), or VEGFR3 AAA -EGFP (R3 AAA ) using lentivirus-mediated gene transfer. After 72 hours, the resulting cells were lyzed and immunoprecipitated with EGFP antibody coupled to the magnetic Dynabeads and immunoblotted using VEGFR3 and CAV-1 antibodies. Immunoblotting of overexpressed VEGFR3-EGFP and VEGFR3 AAA -EGFP (R3/R3 AAA -EGFP) were detected using VEGFR3 antibody. The input lysates were shown on the right. ( C ) VEGFR3 AAA increases VEGFR3 signaling. hLECs were transduced as in ( B ), and the resulting cells were serum starved and treated with 100 ng/ml VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. Quantitative data of VEGFR3 activation ( D ), AKT activation ( E ), and ERK activation ( F ) were shown. n=3 independent repeats. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human dermal lymphatic microvascular endothelial cells (hLECs) (Angio-Proteomie, Cat # cAP-0003) were cultured in Endothelial Cell Medium (ScienCell, Cat # 1001).

Techniques: Binding Assay, Transfection, Control, Immunoprecipitation, Western Blot, Activation Assay