P smad1

Lung tissues were lysed in Tissue Protein Lysis Solution (CWBIOTECH, Beijing). Protein from each sample was loaded into a 10 % sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). The blocked membrane was then incubated with various antibodies. Antibodies against IL-27 (C-8), COL1A2 (M-19), p-Stat1 (Tyr 701), p-Stat5 (Tyr 694/Tyr 699), p-Smad3 (Ser 208), TGF-βR1 (V-22), p-Stat3 (B-7), and p-Smad1 (Ser 465) were purchased from Santa Cruz Biotechnology. Antibodies against SOCS3, SMAD1, STAT3, STAT5, STAT1, SMAD3, and β-actin were purchased from Proteintech. The immunoreactive bands were visualized with an enhanced chemiluminescent (ECL) reagent (Beyotime). The signal intensity of the bands was quantified using Image J software.

Western blot assay was applied to the detection of TGFBR3, total (t)-Smad1, phosphorylated (p)-Smad1, t-Smad3, and p-Smad3 protein in the collected cells.
Total protein of cells was extracted and the protein concentration was determined based on the bicinchoninic acid kit. The protein sample was loaded to the wells in sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with skim milk and incubated with primary antibodies TGFBR3 (1:2000, R&D Systems, Minneapolis, MN, USA), t-Smad1 (1:1000), p-Smad1 (1:1000, Santa Cruz Biotechnology), t-Smad3 (1:1000), p-Smad3 (1:1000), and GAPDH (1:1000, all from Abcam, Cambridge, MA, UK) which was followed by incubation with the horseradish peroxidase-labeled secondary antibody (1:500, Jackson ImmunoResearch Laboratories, PA, USA). Washed 3 times by tris-buffered saline with Tween 20, the membrane was developed by enhanced chemiluminescence. Quantification of signals was completed by the National Institutes of Health ImageJ Imaging. Processing Analysis Software with signaling intensity normalized to GAPDH.