Phosphorylated smad2 3

Harvested cells were washed twice with cold PBS and then lysed using RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail (Selleck, Shanghai, China). The lysates were centrifuged for 15 min at 12,000×g at 4°C and the supernatant was collected. The supernatant was boiled for 5 min and then separated by 10% SDS-PAGE gels. The proteins were transferred to a PVDF membrane (Millipore, Bedford, MA, USA), blocked with 5% milk, and incubated overnight with E-cadherin (Abcam, Cambridge, MA, USA), α-SMA (Abcam), TGF-β1 (Abcam), phosphorylated ERK1/2 (Cell Signaling Technology, Boston, USA), ERK1/2 (Cell Signaling Technology), phosphorylated Smad2/3 (Cell Signaling Technology), Smad2/3 (Cell Signaling Technology) and phosphorylated JNK (Cell Signaling Technology). Membrane was then washed three times in TBST and incubated with peroxidase-labelled secondary antibodies (Boster, Inc., Wuhan, China) for 2 h at room temperature. Following three washes in TBST, bands were visualized by western electrochemiluminescence (ECL) kit (Tanon, Shainghai, China) and then exposed to X-ray film. The band densities were quantified using the AlphaEaseFC image analyzer system (Alpha Innotech, Inc., CA, USA) and the results were expressed as ratio of band density to total GAPDH.

CG200745 was generously donated by Crystal Genomics Inc. (Seoul, Rep. Korea). Anti-rabbit antibodies against TGF-β1 (polyclonal; Cell Signaling Technology, MA, USA), extracellular signal regulated kinases 1/2 (ERK 1/2), anti-phosphorylated ERK (p-ERK 1/2), anti-c-Jun N-terminal kinase (JNK), anti-phosphorylated JNK (p-JNK), anti-total p38, anti-phosphorylated p38 (p-p38), Smad2/3, and phosphorylated Smad2/3 (Cell Signaling Technology, MA, USA), anti-mouse antibodies against GAPDH (clone GAPDH-71.1;monoclonal), α-SMA (1A4 Clone; monoclonal; Sigma Chemical Co. St. Louis, MO, USA), fibronectin (BD Biosciences, San Jose, CA, USA), heme oxygenase-1 (HO-1, Abcam, Inc., Cambridge, MA, USA), and F4/80 (AbD Serotec, Raleigh, NC, USA) were commercially obtained.

Three duplicated wells of C3H10T1/2 cells at passage 3 were prepared for protein extraction. Cell lysates were extracted using a modified radioimmunoprecipitation assay (RIPA) lysis buffer that contained 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Cell Signaling Technology, USA). Following centrifugation at 12000g for 30 minutes, the supernatant was collected to detect the total protein concentrations using a BCA Protein Assay kit (Thermo Scientific, USA). Protein extracts were boiled for 5 minutes in loading buffer. Then, 40 μg protein was loaded and electrophoresed on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in a 5% skim milk solution and incubated with primary antibodies overnight at 4°C. The primary antibodies were β-actin (Sigma, A1978), Smad2/3 (Cell Signaling Technology, #8685), and phosphorylated-Smad2/3 (Cell Signaling Technology, #8828). The membrane was then incubated with a fluorescent secondary antibody (LI-COR, 926-32212) (1/1000), for 1 hour at room temperature (Vector Laboratories, Burlingame, VT, USA). The blots were visualized using a LI-COR Odyssey® scanner (LI-COR Biosciences, USA). The relative density of the p-Smad2/3 bands was normalized to their corresponding actin bands.

Cells were divided into four groups as follows: negative control siRNA (NC siRNA), NC siRNA + BSHX, Tgfβr2 siRNA, and Tgfβr2 siRNA + BSHX. All cells were lysed in lysis buffer to obtain total protein extracts, respectively. After being incubated on ice for 30 min, the extracts were separated with 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were then blocked in 5% milk for 1 h and incubated with different primary antibodies against phosphorylated-SMAD2/3 (diluted 1:1000, Cell Signaling Technology, #8828, USA) and SMAD2/3 (diluted 1:1000, Cell Signaling Technology, #8685, USA) overnight at 4°C. Next day, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (diluted 1:5000; Abcam, ab6721, USA) at room temperature for 1 h. The densities of the bands were visualized by chemiluminescence.