Mechanical force on ICAM-1 increases RhoA activity and MLC phosphorylation. Magnetic beads coated with mAb against ICAM-1 (A, C, and D) or MHC class I (B and C) were added for 15 min to a monolayer of TNF-treated HUVECs and ~10 pN force was applied with a ceramic magnet placed above the cells for 1 min. (A and B) Using GST-RBD, RhoA. GTP was isolated and detected by immunoblotting. (C) Phase contrast images of EC monolayers 15′ after beads were added and washed 2x with media before fixing. (D) Lysates were immunoblotted for total MLC or MLC phosphorylated on Thr18/Ser19. Graphs show quantification of RhoA activity (A and B) or pMLC levels (D) from ≥3 independent experiments. Graphs show the means ± SEM. Asterisk shows p-value of statistical significance compared to control (*, p≤0.05; **, p≤0.01).