P limk1

HUVECs were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA) maintained in vascular cell basal medium (ATCC, Manassas, VA, USA) and used before to passage 7. Fetal bovine serum, phosphate-buffered saline (PBS), penicillin–streptomycin (PS) and 0.25% (w/v) trypsin/1 mM EDTA were all purchased from Invitrogen (Carlsbad, CA, USA). EC growth supplement, heparin, gelatin were supplied by Sigma (St Louis, MO, USA). Dimethyl sulfoxide (DMSO) and anti-VE-cadherin antibody, phospho [Tyr731] were also acquired from Sigma. Antibodies of ve-cadhrin, p-LIMK1, LIMK1, P-MYPT1, MYPT1, P-cofilin, cofilin and beta-actin were all purchased from Cell Signaling Technology (Danvers, MA, USA).

Human lung microvascular endothelial cells (HLMVECs), THP-1 cells, MLE12 cells, and Raw 264.7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HLMVECs were cultured with EGM-2MV microvascular endothelial growth medium containing 5% fetal bovine serum (FBS) at 37 °C in 5% CO₂. LPS (E. coli O55:B5), thrombin, and the anti-β-actin antibody were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against VE-cadherin, USP40, ICAM1, VCAM1, Lamin A/C, and GAPDH and immobilized protein A/G beads were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against pMLC, MLC, the Flag tag, the V5 tag, pLimk1, Limk1, p-cofilin 1, cofilin 1, pIκBα, p65, p-p65, HSP90β, K63-linked ubiquitin, ubiquitin, and acetylated lysine (AcK) were purchased from Cell Signaling (Beverly, MA, USA). ELISA kits for quantifying TNFα, IL-6, and IL-8 were obtained from eBioscience (San Diego, CA), and mouse IL-1β, KC/CXCL1 ELISA kits, and human recombinant TNFα protein were purchased from R&D Systems (Minneapolis, MN, USA). The anti-CD31-FITC antibody was obtained from Biolegend (San Diego, CA, USA). GeneJet™ reagent and GeneMute siRNA transfection reagent were purchased from SignaGen (Frederick, MD, USA). All materials used in the experiments were of the highest grade commercially available.