Image analyzer 680

LECs were pre-cultured with FBS-free and supplement-free EGM2 medium (Lonza) for 24 h. Cells were then treated with CCN2-containing FBS-free and supplement-free EGM2 medium and cultured for the appropriate time. Cells were washed with PBS and lysed in RIPA Buffer (Merck) containing cOmplete protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Nacalai, Kyoto, Japan). Protein concentration was determined with the BCA protein assay kit (Thermo). Protein samples (5–20 µg) were separated with SDS-PAGE and transferred to PVDF membranes (Invitrogen). The membranes were blocked in Blocking-one (Nacalai) for 30 min and cut prior to hybridization with antibodies, then incubated with primary antibodies at 4 °C overnight. The primary antibodies are listed in Supplementary Table S3. After three washes with PBS containing 0.1% Tween-20, the membrane was incubated with HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-rat IgG secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 2 h. After three washes with PBS containing 0.1% Tween-20, signals were visualized using ECL Prime (GE Healthcare, Chicago, IL, USA) and an image analyzer 680 (GE Healthcare). Signals were quantified using ImageQuant TL 8.1 software (GE Healthcare).

LECs were pre-cultured with FBS-free and supplement-free basal medium for 24 h and then treated with medium containing 100 ng/mL of recombinant rat CCN2/CTGF (R&D Systems) for 5 min. The phosphorylation levels of RTK were analyzed using a Proteome profiler mouse phospho-RTK array kit (R&D Systems) according to the manufacturer’s instructions. Briefly, 200 µg of total protein was incubated with the receptor antibody-spotted membrane at 4 °C for 24 h. After washing, the membrane was incubated with anti-phospho tyrosine kinase antibody, and the phosphorylation levels of receptor tyrosine kinase were detected with the image analyzer 680 (GE Healthcare).