cANGPTL4 interacts with integrin α5β1, VE-cadherin, and claudin-5. (A-E) Representative sensorgrams showing binding profiles between immobilized cANGPTL4 and integrin α5β1, VE-cadherin, claudin-5, occludin, and JAM-A (A); EC1, ECD1, VE-cadherin EC3, and claudin-5 ECD2 (B); integrin α5β1 (C), VE-cadherin (D), and claudin-5 (E) preblocked with either preimmune IgG or indicated cognate antibodies. (F) In situ PLA of cANGPTL4 and indicated binding partners. PLA signals are in red (integrin 22.50 ± 5.01 vs VE-cadherin 16.43 ± 2.46 vs claudin-5 12.35 ± 3.44 PLA spots/cell for 250 cells, n = 3). Cells were counterstained with Alexa 488-phallodin for actin stress fibers (green) and Hoechst dye for nuclei (blue). Negative control was performed in the absence of anti-ANGPTL4. Scale bar represents 40 μm. (G) Immunodetection of VE-cadherin and claudin-5 from membrane extract of HMVECs treated with cANGPTL4 (3 or 6 μg/mL) for 3 hours. Vehicle (v) is PBS, and E indicates treatment with 2.5mM EDTA in PBS.