HUVECs were treated with or without 10 nM PMA in M199 containing 5% FBS for 48 h. These cells were harvested using Accutase, suspended in serum-free M199, and plated on fibronectin-coated coverslips for 1 h. Cells were fixed in 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Cells were blocked with 1% BSA and incubated with anti-TM (Dako, Carpinteria, CA, USA) and anti-FAK (Invitrogen) antibodies for 1.5 h at room temperature, followed by incubation with Alexa Fluor 488- or 546-conjugated secondary antibodies (Invitrogen) for 1.5 h at room temperature. Alexa Fluor 555 phalloidin (Invitrogen) was used to stain filamentous actin. Images were taken with an Olympus Fluoview FV1000 confocal laser scanning microscope using a ×63 objective (Olympus, Tokyo, Japan) at identical exposure times (12.5 μs/pixel). For staining of TM and fibronectin during tube formation, HUVECs, which were treated with or without 10 nM PMA in M199 containing 5% FBS for 48 h, were harvested using Accutase, suspended in M199 containing 5% FBS, and plated on Matrigel-coated coverslips for 1.5 or 3 h. Cells were doubly stained with anti-TM (Dako) and anti-fibronectin (F3648, Sigma-Aldrich) antibodies.
Hsu Y.Y., Shi G.Y., Wang K.C., Ma C.Y., Cheng T.L, & Wu H.L. (2016). Thrombomodulin promotes focal adhesion kinase activation and contributes to angiogenesis by binding to fibronectin. Oncotarget, 7(42), 68122-68139.
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