Calponin

Tanshinone IIA (99.0% pure; Boyun Biotech Company, Shanghai, China) was prepared as a 10-mg/ml stock solution in dimethyl sulfoxide and stored in the dark at −20°C. The following antibodies were used for immunofluorescence or Western blotting: collagen 1, collagen 3 and α-smooth muscle action (α-SMA) (Abcam, New York, USA). Myosin and calponin were purchased from Proteintech Group, Inc. (Chicago, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), p38, phospho-p38, Jun N-terminal kinase (JNK), phospho-JNK, extracellular signal-regulated kinase (ERK) and phospho-ERK were obtained from Cell Signaling Technology (Boston, USA). Smooth muscle cell medium-basal was obtained from Sciencell (San Diego, California, USA). Human recombinant IL-17A was purchased from R&D Systems (Minneapolis, Minnesota, USA). Other chemicals and materials were obtained from the Beyotime Institute of Biotechnology or KeyGEN Biotechnology Company (Nanjing, China).

An antibody against TGF-β1 (cat. no. BA0290) was purchased from Boster Biological Technology, Ltd. (Wuhan, China). Antibodies against Smad2 (cat. no. 5339), phosphorylated (p)-Smad2 (Ser465/467; cat. no. 3108), Smad3 (cat. no. 9523), p-Smad3 (Ser423/425; cat. no. 9520), extracellular signal-regulated kinase (ERK; cat. no. 4695) and p-ERK (Thr202/Tyr204; cat. no. 4370) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against Smad4 (cat. no. D120124), Smad7 (cat. no. D160746), p38 (cat. no. D151619) and p-p38 (Thr180/Tyr182; cat. no. D151619) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Antibodies against proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and calponin were purchased from ProteinTech Group, Inc. (Chicago, IL, USA). Horseradish peroxidase (HRP)-conjugated goat-anti rabbit (cat. no. A0208), goat anti-mouse (cat. no. A0216) IgG secondary and Cy3-labeled secondary antibodies (cat. no. A0516) were obtained from Beyotime Institute of Biotechnology. Antibody against internal reference protein β-actin were purchased from Bioss (Beijing, China).

A bicinchoninic acid (BCA) protein kit from Abcam in the United States was used to measure the protein concentrations after the tissues were processed with RIPA lysis buffer (Beyotime, China). Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 8% or 12%, the proteins were separated before being transferred to a polyvinylidene fluoride (PVDF) membrane (Beyotime, China). The PVDF membrane was incubated with the following primary antibodies overnight at 4 °C after being blocked with 5% skim milk for 1 h：GAPDH (1:10,000, Cat. No. 60004-1-Ig, Proteintech), α-SMA (1:1,000, Cat. No. 14395-1-AP, Proteintech) and Calponin (1:1,000, Cat. No. ab46794, Abcam) [49 ,50 (link)].