α sma 14395 1 ap

The following primary antibodies were used for western blotting, immunofluorescence, and immunohistochemical analysis: p-c-Met (#3077; Cell Signaling Technology, Danvers, MA, USA), c-Met (ab51067; Abcam, Cambridge, UK), p-mTOR (#2971; Cell Signaling Technology), mTOR (#2983; Cell Signaling Technology), p-p44/42 MAPK (#9101; Cell Signaling Technology), p44/42 MAPK (#9102; Cell Signaling Technology), α-SMA (14,395-1-AP; Proteintech, Rosemont, IL, USA), MMP2 (10,373-2-AP; Proteintech), MMP9 (10,375-2-AP; Proteintech), TIMP2 (sc-21735; Santa Cruz Biotechnology, Dallas, Texas, USA), COL I (immunofluorescence: ab6308; Abcam; immunohistochemistry: LS-C343921; LSBio, Seattle, WA, USA), COL III (immunofluorescence: ab7778; Abcam; immunohistochemistry: LS-C413514; LSBio), β-actin (sc-47778; Santa Cruz Biotechnology), VEGF (sc-57496; Santa Cruz Biotechnology), and vimentin (ab92547; Abcam). The following secondary antibodies were used: anti-rabbit IgG HRP (western blotting: #7074; Cell Signaling Technology: immunofluorescence: R37117; Thermo Fisher, Waltham, MA, USA; immunohistochemistry: PK-4001; Vector Laboratories, Burlingame, CA, USA) and goat anti-mouse IgG HRP (western blotting: ADI-SAB-100; Enzo Life Science, Farmingdale, NY, USA; immunofluorescence: A11005; Thermo Fisher; immunohistochemistry: PK-4002; Vector Laboratories).

Total protein was extracted from mouse left lung tissue, and its concentration was determined using the bicinchoninic acid assay. Then the constituent proteins were separated by electrophoresis in a 10% sodium dodecyl sulfate polyacrylamide gel, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States), covered with 5% milk at room temperature for 1 h, and incubated overnight with appropriate primary antibodies diluted in 0.1% Tween 20 (TBST) at 4°C. Primary antibodies against the following proteins were used: SMAD2/3 (8685T, Cell Signaling Technology, Beverly, MA, United States), p-SMAD2/3 (8828S, Cell Signaling Technology), TGF-β1 (3711S, Cell Signaling Technology); GAPDH (AC002, Abclonal, Wuhan, China); αSMA (14395-1-AP, Proteintech), collagen I (14695-1-AP, Proteintech), and PAI-1 (TA504056S, Origene, Maryland). After washing with TBST, the membranes were incubated with anti-rabbit or anti-mouse IgG horseradish peroxidase conjugated antibody (Cell Signaling Technology) for 1 h at room temperature. The protein bands were visualized using SuperSignale West Pico plus Chemiluminescent Substrate (Thermo Fisher Scientifice, Waltham, MA, United States).

Total proteins from liver homogenates were extracted using RIPA buffer. Nucleus and cytoplasm proteins were prepared using the extraction buffer as previously described[18 (link)]. Identical amounts of proteins were subjected to SDS-PAGE, and then transferred to PVDF membranes. The membranes were blocked with 5% milk, and then incubated with following primary antibodies: FAS (H-300, #sc-20140), ACCα (H-76, #sc-30212), SREBP1 (H-160, #sc-8984), COL4A2 (T-15, #sc-70246), TGFβ1 (V, #sc-146), MCP1 (FL-148, #sc-28879) and Nrf2 (C-20, #sc-722) from Santan Cruz Biotechnology, USA; IL-1β (#16806-1-AP), TNFα (#60291-1-Ig) and αSMA (#14395-1-AP) from Proteintech, USA; Histone H3 (T22, #BS1405) from Bioworld Technology, USA; β-actin (1E9A3, #TA-09) from ZSGB-BIO, China. Thereafter, the membranes were incubated with corresponding secondary peroxidase-coupled antibody. Finally, the detection procedure was performed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Temecula, CA, USA). Densitometric analysis was performed using the Image J software. β-actin served as a loading control for total or cytosolic proteins, and Histone H3 served as the loading control for nuclear proteins.