Ab40854

The relevant protein levels of TGF-β1, Smad3, P-Smad3, Smad7, α-SMA and collagen-1 in vitro were measured by western blot. Cells incubated and treated in 6-well plates were lysed by 200 μL RIPA lysis buffer containing protease inhibitor (PMSF, 1 mM) and phosphatase inhibitor (TIANDZ 80809-1, 1%) on ice-bath for 30 min. And the lysate was centrifuged at 10000 rpm for 10 min at 4 °C, the supernatant was collected and the proteins were quantified by BCA protein assay kit (Beyotime Biotechnology, China). Membranes were incubated with primary antibodies (anti-TGF-β1, 1:1000, Abcam, ab64715; anti-Smad3, 1:1000, Abcam, ab40854; anti-P-Smad3, 1:400, Bioss, BS-3425R; anti-Smad7, 1:400, Boster, BA1399; anti-α-SMA, 1:400, Boster, BM0002; anti-collagen1, 1:400, Boster, BA0325) overnight at 4 °C and mouse monoclonal anti-β-actin (1:500, Sinipept, 41302M) was used for loading control. After that HRP-goat-anti-mouse antibody (1:1000, World Biotech, WH-004) was incubated with membranes of TGF-β1, α-SMA and β-actin for 1 h at room temperature; HRP-goat-anti-rabbit antibody (1:1000, CW-biotech, cw-0103) with Smad3, P-Smad3, Smad7 and collagen-1 respectively. Immunoblot were visualized with Bio-Rad and recorded by Bio-Rad detection system, and gray density analysis was completed with the Image J program.

Western blotting was conducted using standard procedures. Total protein was isolated from the treated fibroblasts and tracheal tissues using radioimmuneprecipitation assay buffer (high)-phenylmethylsulfonyl fluoride (RIPA-PMSF; SolarBio, Beijing, China). Subsequently, total protein content was determined by BCA assay kit (SolarBio, Beijing, China). An equal number of proteins were electrophoresed through SDS-PAGE, and then transferred onto nitrocellulose membranes (Merck Millipore, Germany). After inhibiting with 5% skimmed milk for 2 h, the membranes were incubated at 4°C for 12 h with the following primary antibodies: α-SMA (E-AB-34,268; Elabscience, China), COL1 (collagen 1; ab34710), Akt (ABP50629; Abbkine, USA), p-Akt (ab19623), mTOR (ab32028), p-mTOR (ab109268), Smad2 (1:2000, ab40855), p-Smad2 (ab188334), Smad3 (ab40854), p-Smad3 (ab52903), TGF-β1 (ab179695) and β-actin (1:5000, bs-0061 R; Bioss, China). All antibodies were purchased from Abcam (USA), with a dilution of 1:1000, unless otherwise stated. After rinsing 3 times in TBST, the membranes were incubated with the respective secondary antibodies at ambient temperature for 2 h. Lastly, the immunoblots were detected by electrochemiluminescence plus reagent kit (Biosharp, Guangdong, China), and densitometric analysis of each target protein band was conducted using ImageJ software (NIH).