Ecl western blotting detection system

Protein samples were separated by SDS–polyacrylamide gel electrophoresis and blotted on polyvinylidenfluoride membranes (Merck Millipore). Membranes were blocked overnight with Tris-buffered saline 1X (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 8) containing, 0.1% Tween-20 and 8% dry milk and then incubated for an additional 3 hr with the primary antibodies diluted in TBS 1X, 0.1% Tween-20, 5% dry milk. The different polyclonal antisera to ZitP (NTER, 1:5000 dilution and CTER, 1:5000), CtrA (1:10,000) and DivJ (1:10,000) were used. Commercial and polyclonal antibodies to Dendra2 (Antibodies-Online) and mCherry (Chen et al., 2005 (link)) were used at 1:5,000 and 1:10,000 dilutions respectively. Primary antibodies were detected using HRP-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) with ECL Western Blotting Detection System (GE Healthcare) and a luminescent image analyzer (Chemidoc^Tm MP, Biorad).

Collagen was purchased from Takeda Austria GmbH (Linz, Austria). Rivaroxaban and edoxaban were kindly provided by Bayer Vital GmbH (Leverkusen, Germany) and Daiichi Sankyo Co., Ltd. (Tokyo, Japan), respectively. Phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific HSP27 antibodies (Ser-78) and phospho-specific HSP27 antibodies (Ser-82) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). HSP27 antibodies and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The PDGF-AB enzyme-linked immunosorbent assay (ELISA) kit was obtained from R&D System, Inc. (Minneapolis, MN). The phosphorylated HSP27 ELISA kit was purchased from Enzo Life Science INC. (Farmingdale, NY). ECL Western blotting detection system was obtained from GE Healthcare (Buckinghamshire, UK). Other materials were obtained from commercial sources.

Western blot analysis was performed as previously described 52 (link), 53 (link), 54 (link).Protein lysates/extracts (40 μg) were prepared from the BMMSCs, ECs, and exosomes using RIPA lysis buffer (Thermo Fisher Scientific) and loaded on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) independently. Separated proteins in the gels were transferred to polyvinylidene difluoride (PVD) membranes using an electrotransfer apparatus (Bio-Rad, USA). Following blocking the membrane with 5 % non-fat dry milk (1 h), the membranes were probed with a primary antibody (anti-RUNX2 (ab23981), anti-Bglap (ab13420), anti-Tie-2 (ab111074) and anti-phosphor Tie2 (Y992, ab151704), anti-NOS3 (ab76198) and anti-phosphor NOS3 (S1177, ab230158), anti-CD9 (ab223052), anti-CD63 (ab8219)) for 2 h at 4 °C. Then membranes were further incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were developed with the ECL Western blotting detection system (GE Healthcare, Piscataway, NJ, USA) and the image was recorded using a gel documentation system (Bio-Rad, USA). Each target protein band density was normalized with a respective GAPDH density using Image Lab densitometry software (Bio-Rad) and expressed as fold changes to control.

Cell lysates were suspended in RIPA buffer (50mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% SDS, 1mM Na₃VO₄, 2mM EDTA, 1mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin) and incubated for 1 h at 4°C. After electrophoresis, proteins were transferred onto a nitrocellulose membrane (Hybond-ECL, GE Healthcare). Membranes were incubated with anti-Smad3 (Abcam), pSmad3 (Cell Signaling Technology), α-tubulin, Elf3, RII, and WT1 (Santa Cruz Biotechnology) antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). The immunoreactive bands were visualized using an ECL Western blotting detection system (GE Healthcare).