Ecl plus blocking agent

In Western Blot analysis, whole cell lysate was prepared from primary culture of macrophages treated with indicated stimuli in the Results or Figure Legends by RIPA buffer (Sigma Aldrich, St. Louis, MO) supplemented with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). After Sodium Dodecyl Sulfate- Polyacrylamide gel Electrophoresis (SDS-PAGE), separated proteins were transferred to a PVDF membrane (Immobilon-P, Merck) and blocked with an ECL plus blocking agent (GE healthcare, Buckinghamshire, UK). Blotted membranes were, then, incubated with primary antibodies followed by incubation with anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by the chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
Primary antibodies used in this study are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (ser536) antibody (Cell Signaling Technology), mouse monoclonal anti-IκBα antibody (Cell Signaling Technology), mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).

Primary culture of SMCs was treated with 100 ng/ml recombinant PDGF-BB (R&D Systems) or 100 ng/ml AREG (R&D Systems) for 72 h. Whole cell lysate was then prepared by a RIPA buffer (Sigma Aldrich, St. Louis, MO) supplemented with proteinase inhibitors and phosphatase inhibitors (Roche). Protein concentration was determined by a bicinchoninic acid (BCA) method (Pierce BCA Protein Assay Kit, Thermo Scientific). After Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE), separated proteins were transferred to a PVDF membrane (Hybond-P, GE healthcare, Buckinghamshire, UK) and blocked with an ECL plus blocking agent (GE healthcare). Membranes were then incubated with primary antibodies followed by incubation with anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by a chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
The following primary antibodies were used: mouse monoclonal anti-SMA antibody (#M0851, Dako) and mouse monoclonal anti-α-tubulin antibody (#T6199, Sigma Aldrich).

Whole cell lysate was prepared by a RIPA buffer (Sigma Aldrich) supplemented with proteinase and phosphatase inhibitors (Roche, Indianapolis, IN). Protein concentration was, then, determined by a bicinchoninic acid (BCA) method (Pierce BCA Protein Assay Kit, Thermo Scientific, Waltham, MA). After sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), separated proteins were transferred to a PDVF membrane (Hybond-P, GE healthcare, Buckinghamshire, UK) and blocked with an ECL plus blocking agent (GE healthcare). The membranes were incubated with primary antibodies followed by incubation with an anti-IgG antibody conjugated with horseradish peroxidase (GE healthcare). Finally, the signal was detected by a chemiluminescent reagent (ECL Prime Western Blotting Detection System, GE healthcare). α-Tubulin was served as an internal control.
Antibodies used in Western blot analysis are as follows: rabbit monoclonal anti-p65 antibody (Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-phospho NF-κB p65 (Ser536) antibody (Cell Signaling Technology), and mouse monoclonal anti-α-tubulin antibody (Sigma Aldrich).