Sc 390268

Primary antibodies against pDDR1-Y792(11994S; CST; 1:1000), DDR1 (10536-1-AP; Proteintech; 1:1000), YAP1 (13584-1-AP; Proteintech; 1:1000), pYAP1-S127 (AP0489; ABclonal; 1:1000), YWHAE (11648-2-AP; Proteintech; 1:1000) VE-cadherin (bs-4310R; Bioss; 1:1000) and GAPDH (BE0024; Easybio; 1:4000) were used for Western blotting. Rhodamine Phalloidin (RM02835; ABclonal; 1:200), Primary antibodies against YAP1 (66900-1-Ig; Proteintech; 1:200), DDR1 (10536-1-AP; Proteintech; 1:200), VE-Cadherin (sc-9989; Santa Cruz; 1:100), E-selectin (MA1-22165; Invitrogen; 1:200), ICAM1 (sc-7891; Santa Cruz; 1:100), VCAM1 (sc-13160; Santa Cruz; 1:100), Caveolin-1 (66067-1-Ig; Proteintech; 1:200), Rab 5A (sc-166600; Santa Cruz; 1:100), PE-conjugated CD63 (BC-353003; BaiCheng Technology; 1:200) and LATS1 (3477S; CST; 1:200) were used for immunofluorescence staining. DDR1 antibody (200 μg/ml; sc-390268; Santa Cruz) were used for bead pulling/magnetic tweezer system. The validation of all primary antibodies for the species and application were described on the manufacturer’s website.

HUVECs were cultured in 33 mm glass bottom dishes to form a sub-confluent monolayer. After the cells were fully attached, 4 µM of Fluo-4 AM calcium binding dye (F14217; invitrogen) was added to the media. Cells were incubated with the dye for 30 min. Beads conjugated with either collagen or DDR1 antibody (sc-390268; Santa Cruz) were added and the cells were incubated for another 30 min. To assess the calcium influx as a result of mechanical stimulation, the cells with Fluo-4 and magnetic beads were subjected to a force of 4 pN applied with magnetic tweezer. Time-lapse video of calcium imaging was acquired with the total internal reflection fluorescence (TIRF) microscope for 10 s before stimulation, 120 s stimulation and 30 s after stimulation. Acquired image sequences were analyzed by measuring the mean fluorescence intensity (mean pixel value) of each cell at each frame. Mean peak amplitude was calculated and normalized to the pre-stimulation fluorescence intensity for each cell.