Ab182973

Proteins isolated from cultured cardiac myocytes and mouse hearts were determined by Western blot analysis. Equal amounts of protein were subjected to SDS-PAGE. A standard Western blot analysis was conducted using serpine1 antibody (1:1,000 dilution; ab182973, Abcam). β-Tubulin antibody (1:2,000 dilution; 2128S, Cell Signaling Technology) was used as the loading control. A cooling charged coupled device (CCD) imaging apparatus (Tanon-5200) was used for image capture.

Total proteins were extracted from cells using RIPA buffer (Beyotime Institute of Biotechnology). The proteins were separated by 10% SDS‒PAGE and transferred to a PVDF membrane (EMD Millipore). The membrane was blocked with 5% fat-free milk and incubated with primary monoclonal rabbit anti-ETV7 (1:1000, ab229832, Abcam), mouse anti-ETV7 (1:500, E-1, Santa Cruz), rabbit anti-BATF2 (1:500, 1B11, Santa Cruz), rabbit anti-PAI-1 (1:500, ab182973, Abcam), rabbit anti-phospho-ERK1/2 (1:1000, 4370, CST), rabbit anti-ERK1/2 (1:1000, 4695, CST) and mouse anti‐GAPDH (1:3000, TA-08, ZSGB-BIO) for immunoblotting. The ECL detection system (EMD Millipore) was used to assess protein expression. GAPDH was used as the internal control. The experiments were conducted in triplicate.

For protein analysis, BEAS-2B cells were plated in 6 well culture dishes and treated with either MWCNT (5 μg/mL) and/or TGF-β for 72 h. Cells were lysed with RIPA buffer containing Halt^TM Protease and Phosphatase inhibitor cocktail (Cat#: 78440, Thermo Fischer Scientific, Waltham, MA, USA). Protein concentrations were measured in whole cell lysates by Pierce BCA Protein Assay Kit (Cat#: 23225, Thermo Fischer Scientific, Waltham, MA, USA). Total protein (20 µg) was resolved in a gradient SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with anti-Fn (ab2413, 1:1000, Abcam), anti-PAI-1 (ab182973, 1:1000, Abcam), anti-p21 (ab109199, 1:1000, Abcam), anti-p16 (ab211542, 1:1000, Abcam), anti-γH2A.X (ab2893, 1:1000, Abcam), anti-H2A.X (ab11175, 1:1000, Abcam), anti-E-cadherin (ab11472, 1:1000, Abcam), and anti-vimentin (ab92547, 1:1000, Abcam) primary antibodies overnight at 4 °C. Membranes were incubated with HRP-conjugated secondary anti-rabbit (Cat#1706515, 1:5000, BioRad, Hercules, CA, USA), or anti-mouse (Cat#: 1706516 1:5000, BioRad, Hercules, CA, USA) for chemiluminescence detection using the Bio-Rad ChemiDoc MP imaging system. Image Lab software (v4.1, BioRad, Hercules, CA, USA) was utilized for densitometric quantification of band intensities.