Active rac gef assay kit

Assays were performed using the active Rac-GEF assay kit (Cell Biolabs, San Diego, USA) according to the manufacturer’s instructions. Briefly, cells were washed twice with ice-cold PBS and lysed in ice-cold 1 × Assay/Lysis Buffer (1 mM PMSF, 10 μg ml⁻¹ leupeptin and 10 μg ml⁻¹ aprotinin). Extracts were incubated with 40 μl of resuspend Rac1 G15A agarose bead slurry and incubate for 1 h at 4 °C. Beads were washed three times with the 1 × Assay/Lysis Buffer, resuspended in 40 μl of 2 × reducing SDS–PAGE sample buffer and boiled for 5 min. Pull-down supernatant were subjected to SDS–PAGE electrophoresis and western blotting with anti-Tiam1 antibody.

A549, H460 (American Type Culture Collection, USA), H1299 and H23 cells (kindly provided by Dr Jin Yang, Huazhong University of Science and Technology) were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Gibco, USA). Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical veins of fresh cords. The cells were maintained in Endothelial Cell Medium (Sciencell, USA). A total of 61 paired specimens (adjacent tissues and tumor) were collected immediately following lobectomy of LuAC patients at the Union Hospital (Wuhan, China). Samples used in this study were approved by the Committees for Ethical Review of Research at Huazhong University of Science and Technology. Par3 (ab64646), Rac1 (ab155938) and CD31 (ab28364) antibodies was purchased from Abcam (Abcam, USA); 14-3-3ζ (sc-1019), anti-pJAK2^Y1007/8 (sc-21870), anti-JAK2 (sc-278), anti-pSTAT3^Y705 (sc-7993), anti-ZO-1 (sc-10804), and anti-STAT3 (sc-482) were from Santa Cruz Biotechnology (Santa Cruz, USA); and anti-Tiam1 (STA-422) was from the Active Rac-GEF assay kit (Cell Biolabs, USA).