Fig. 4

VEGFR2 specifically associates with Sdc2 upon VEGFA₁₆₅ stimulation. a–f Mouse ECs (a) or HUVEC (b–f) were transduced for 16 h with adenovirus expressing the indicated construct (MOI = 1–2), starved for 8 h and then stimulated with VEGFA₁₆₅ (50 ng/ml), VEGFA₁₂₁ (50 ng/ml), or FGF2 (20 ng/ml). Anti-HA pulled down (IP) was performed for 2 h at 4 °C followed by western blot analysis to check co-immunoprecipitated proteins (WB). Whole-cell lysates (lysate) were analyzed for total protein levels. b HS digestion with Heparinases or K5 lyase (1 h at 37 °C) before VEGFA₁₆₅ cell stimulation prevented formation of VEGFR2–Sdc2 complex. c VEGFA₁₂₁, which lacks heparin-binding domain, was unable to promote VEGFR2–Sdc2 association. d FGF2 did not promote complex formation between Sdc2 and VEGFR2. e Other syndecans displayed minimal or no association with VEGFR2 with or without VEGFA₁₆₅. Red arrows indicate syndecans core in dimeric form with following MW (calculated with signal peptide): Sdc1 ~65, Sdc2 ~44, Sdc3 ~91, Sdc4 ~44. f A chimera construct expressing Sdc2 extracellular domain with Sdc4 transmembrane + intracellular domain (Sdc2^EX Sdc4^IN) showed same extent VEGFA-induced association with VEGFR2 as full length Sdc2. g, h Rescue of VEGFR2 activation is shown with chimera construct Sdc2^EX/Sdc4^IN but not Sdc4^EX/Sdc2^IN (g representative picture, h quantification) (n = 3–4). Errors bars represent SEM. (N.S. not significant, ^**P < 0.01, by one-way Anova with Bonferroni’s multiple comparison test)