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Cells were harvested and fixed in PBS containing 4% of formaldehyde and permeabilized with ice-cold (−20°C) methanol. Cells were blocked in PBS containing 0.1% Tween and 5% BSA; cells were incubated with primary antibodies Pancytokeratin (MA5-13156, Pierce Antibodies, 1:100 dilution), β-catenin (9582, Cell Signaling, 1:100 dilution), Occludin (33-1500, Life Technologies, 1:100 dilution), JAM-A (sc-25629, Santa Cruz, 1:100 dilution), MMP-9 (ab38898, Abcam, 1:100 dilution) overnight at 4°C. Cells were subsequently washed and incubated with secondary antibodies (goat anti-rabbit Alexa Fluor568, 1:500 dilution, and goat anti-mouse Alexa Fluor647, 1:500 dilution; Invitrogen) for 1 hour at room temperature. DAPI (0.5 μg/mL) was used to stain the nuclei. Prolong gold was added to preserve the fluorescent signal and to fix the slides. Pictures were analyzed with Imaris software.

Cells were harvested and solubilized in cell lysis buffer (Cell Signaling, Beverly, MA, USA) for 30 min at 4 °C. Protein concentration was measured using a Bradford protein assay kit (BioRad, Hercules, CA, USA). The same amounts of proteins from whole cell lysates were subjected to SDS polyacrylamide gel electrophoresis and transferred onto methanol-treated PVDF membranes (Millipore Co, Bedford, MA, USA). The membrane was immunoblotted with antibodies against phospho-Akt, VEGF, JAM-A (Santa Cruz, CA, USA), phospho-PKCβ, ZO-1 (Abcam, Cambridge, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA), respectively, overnight at 4 °C. The membranes were washed and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies. After extensive washing, the bands were detected using an enhanced chemiluminescence (ECL) kit (Santa Cruz).

Cells were lysed in RIPA buffer. Protein mixed with loading buffer and heated at 70°C for 10 minutes were loaded on SDS-polyacrylamide gels at 30 ug per lane. The proteins were transferred to PVDF (Millipore, Massachusett, USA) after electrophoresis. Membranes were blocked for 2 hours in 5% bovine serum albumin (BSA) and incubated overnight at 4°C with the SP rabbit polyclonal antibody against E-Cadherin (Sigma), α-Catenin (Sigma), Fibronectin (Cell signaling), Vimentin (Santa Cruz), JAMA (Santa Cruz), Akt (Cell signaling), ERK (Santa Cruz) and GAPDH (Santa Cruz). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000, Santa Cruz Biotechnology) for 1 hour at room temperature. Finally, bands were visualized by enhanced chemiluminescence (Thermo Scientific Pierce, Illinois, USA).

Total protein was extracted from samples of hearts, lungs, and gastrocnemius muscles in RIPA buffer (Sigma-Aldrich, St. Louis, MO) and quantified using a Pierce BCA assay (ThermoFisher Scientific, Waltham, MA). Proteins were separated on 10% Bis-Tris NuPAGE gels in MOPS buffer (ThermoFisher) and transferred to Immobilon-P PVDF membranes (Millipore-Sigma). Blots were washed with Tris-buffered saline (TBS)-Tween 20 (10 mMTris, 0.9% NaCl, 0.1% Tween 20, TBS-T), blocked for non-specific binding with 5% nonfat milk in TBST, and incubated with primary antibodies to specific proteins, β-actin (Sigma-Aldrich A2668), ZO-1, occludin, claudin, and JAM-A (Santa Cruz Biotechnology, sc33725, sc133256, sc81796, and sc53624). Blots were washed in TBST and incubated with secondary antibodies (Cell Signaling Technology 7074, 98,164, and 91,196) linked to horseradish peroxidase (HRP). Protein bands were visualized using enhanced chemiluminescence (100 mM Tris pH 8.6, 0.2 mM p-coumaric acid, 1.25 mM luminol) documented with a FluorChemE imager (ProteinSimple, SanJose CA).

Crystal violet was purchased from Sigma (#3886). The SR-3029 inhibitor was obtained from Glixx Laboratories Inc. Antibodies for CSNK1D (#sc-55553), CSNK1E (#sc-25423), Occludin (#sc-133256), JAM-A (#sc-53623) and GAPDH (#sc-365062) were from Santa-Cruz. Claudin-1 antibody (#13255), horseradish-peroxidase-conjugated anti-rabbit (#7074) and anti-mouse-IgG (#7076) were from Cell signaling.