Anti car

We observed CAR and endothelial cell-to-cell junctional protein expression level; protein was extracted from both WT and KO endothelial cells. Immunoblot analysis used an ECL detection system as described previously [24 (link)]. Primary antibodies were used; anti-CAR (Santa Cruz Biotechnologies, Dallas, TX, USA), PECAM-1, VE-cadherin (R&D system, Minneapolis, MN, USA), FLK-1 (Sigma-Aldrich, Inc. St. Louis, MO, USA), integrin-β1D (Zymed Laboratories, Inc. South San Francisco, CA, USA), and GAPDH (Santa Cruz Biotechnologies, Dallas, TX, USA). Protein expression levels were evaluated with Image Lab software (Bio-Rad laboratories, Hercules, CA, USA) based on the signal intensity of each protein using GAPDH as an internal control for protein loading.

We determined endogenous protein–protein interactions between CAR and the mechanosensory complex (VEGFR2, VE-cadherin, and PECAM-1) using Duolink in situ fluorescence reagents (Sigma-Aldrich). HUVECs adhered to coverslips were transfected with siRNA against CAR for 48 h or exposed to OSS for 24 h and fixed with 2% paraformaldehyde at room temperature for 20 min. After washing with PBS, cells were blocked with 5% normal donkey serum (in 0.5% Triton X-100) for 30 min. Cells were stained with anti-CAR (1:50; Santa Cruz Biotechnology), anti-VEGFR2 (1:50; Cell Signaling Technology), anti-VE-cadherin (1:50; Santa Cruz Biotechnology), or anti-PECAM-1 (1:50; Santa Cruz Biotechnology) antibody. After washing with 0.2% BSA in PBS, coverslips were stained with Duolink probes per the manufacturer’s instructions (Sigma-Aldrich), and probe-conjugated secondary antibodies were added and incubated for 1 h at 37 °C. Next, ligation was performed using the provided ligase for 30 min, followed by amplification using the provided polymerase for 100 min at 37 °C. Nuclei were counterstained with DAPI (100 ng/mL; Santa Cruz Biotechnology) for 5 min. We detected protein–protein interactions between CAR and the mechanosensory complex using a Zeiss LSM 880 Airyscan confocal microscope.