Icam2

Immunofluorescence staining was performed on cell monolayer, 2 hours after the scratch/wounds was performed. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 minutes and permeabilized in 0.1% Triton X-100 (Sigma) for 5 minutes. After blocking with 5% horse serum (Vector Laboratories) for 1 hour, cells were incubated with Alexa Fluor® 488 mouse anti-GM130 antibody (12.5 μg/ml; BD Biosciences) or ICAM2 (0.56 μg/ml; Cell Signaling) for 1 hour. Filamentous actin was stained using Phalloidin conjugated to the fluorescent dye tetramethylrhodamine (TRITC) (1:40; Life Technologies). Samples were mounted using Prolong Gold with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) and imaged with a C2 confocal microscope (Nikon).
Whole mount staining was performed on fibrin gels upon overnight fixation in 4% paraformaldehyde at 4 °C. Immunofluorescence was performed using primary antibodies against Podocalyxin (10 μg/ml; R&D Systems), Collagen IV (5 μg/ml; eBioscience) followed by Texas Red and FITC -conjugated secondary antibodies (1:200, Vector Laboratories). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1000, Life Technologies) and imaged Nikon A1R LUN-V Inverted Microscope.

Cells were washed with PBS then lysed using radioimmunoprecipitation assay (RIPA) buffer (Boston Bioproducts) with protease inhibitor and phosphatase inhibitor cocktail (Roche). The protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientifc). Cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane (Immobilon-P, Millipore), and then blocked in 5% nonfat dried milk for 1 hour. The membrane was then analyzed by immunoblotting with antibodies against the following: phospho-c-ABL (Y245) (0.4 μg/ml; Cell Signaling), c-ABL (1.5 μg/ml; Cell Signaling), phospho-TIE2 (0.002 μg/ml; Cell Signaling), TIE2 (1 μg/ml; Abcam), phospho-AKT (Ser473) (0.05 μg/ml; Cell Signaling), AKT (0.04 ug/mL; Cell Signaling), Podocalyxin (1 ug/mL; R&D Systems), ICAM2 (0.056 μg/ml; Cell Signaling) and β-Actin (1 μg/ml; Sigma). Membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000 Vector Laboratories). Antigen-antibody complexes were visualized using Immobilon Forte Western HRP Substrate (Millipore) on a ChemiDoc™ Gel Imaging System (Biorad) or chemiluminescent sensitive film.

To extract cellular proteins, a lysis buffer (Thermo Fisher Scientific) was employed. A BCA protein assay kit (Beyotime) was used to determine the protein concentration. Subsequently, equal amounts of the protein were separated by SDS-PAGE, and then transferred onto polyvinylidene difluoride membranes. These membranes were blocked using 5% skimmed milk and incubated overnight at 4 ℃ with primary antibodies targeting specific proteins, including ICAM2 (Cell Signaling Technology), GAPDH (Proteintech), BAX (ZENBIO), BAD (ZENBIO), BCL2 (Proteintech), E-cadherin (Proteintech), N-cadherin (ZENBIO), Claudin 1 (Proteintech), Snail1 (Proteintech), B-catenin (Abmart), HA (Proteintech), and RDX (ZENBIO). After washing, the membranes were incubated with secondary antibodies (Proteintech) for 1 h at room temperature. They were then visualized using an enhanced chemiluminescence reagent (Meilunbio) and captured using a ChemiDoc Touch imaging system (Bio-Rad).