Fig. 4

Effect of CAV-1 on VEGFR3 signaling.

a Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). The conserved amino acids are shown in blue. b Co-immunoprecipitation of endogenous VEGFR3 and CAV-1 in hLECs. Lysates from two 10 cm confluent plates of hLECs were combined, then equally divided for immunoprecipitation using VEGFR3 antibody or control protein A beads. The samples were subsequently immunoblotted for CAV-1 and VEGFR3. c, d VEGFR3^AAA loses its binding to CAV-1. c hLECs were transfected with control EGFP, VEGFR3-EGFP (R3), or VEGFR3^AAA-EGFP (R3^AAA) using lentivirus-mediated gene transduction. After 72 hours, the resulting cells were lysed and immunoprecipitated with GFP antibody conjugated to agarose beads and immunoblotted using GFP and CAV-1 antibodies. d The input lysates were immunoblotted using GFP, CAV1, or GAPDH antibody as indicated. e Localization of VEGFR3 and VEGFR3^AAA in caveolae. hLECs were transduced with VEGFR3-APEX2 or VEGFR3^AAA-APEX2 Lenti-viral particles, and after 72 h, cells were fixed with 2.5% glutaraldehyde, stained using DAB substrate kit, and pelleted for TEM analysis. An enlarged view of a single caveola, highlighted with a white box, is shown in the top left corner of each image. f–h hLECs were transduced using lentivirus, and the resulting cells were growth factor starved and treated with 100 ng/mL VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. R3/R3^AAA-EGFP denotes detection using GFP antibody. Quantitative data of VEGFR3 activation (g), ERK activation (i), and AKT activation ( j) were shown. Mean ± SD; two-way ANOVA with Tukey’s post-hoc test. n = 3 independent repeats in g, i, j. Endg: endogenous. Scale bar: 400 nm. Source data are provided as a Source Data file.