Anti col3a1

Soluble proteins from tissues were obtained by overnight shaking (600 RPM) in 0.5 M NaCl, 10 mM Tris Base pH 7.5, 1× protease inhibitor buffer at 4 °C. After centrifugation at 13,000 RPM for 1 min, the supernatants (soluble proteins) were collected and quantified. Samples (10 γg) were separated in NuPAGE™ 4–12% Bis–Tris Midi Protein Gels (Life technologies, Thermo Fisher Scientific, Waltham, MA, USA) by SDS-PAGE with MOPS running buffer (Life technologies) and electroblotted onto nitrocellulose (GE, Healthcare, Little Chalfont, UK) membranes. The blots were incubated with primary and secondary antibodies. The antibodies were revealed using Luminata™ Western HRP Chemiluminescence Substrates (Millipore Corporation, Billerica, MA 01821 U.S.A). To control for equal protein loading and transfer, the membranes were stained with Ponceau S solution (Sigma, St. Louis, MO, USA). The following antibody was used: anti-COL3A1 (Santa Cruz Biotechnology, Dallas, Texas, USA, sc-8781). The secondary anti-rabbit peroxidase-conjugated antibody was from Jackson ImmunoResearch (Cambridge House, UK). The chemiluminescent blots were imaged with the ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

Cardiac fibroblasts/myofibroblasts were lysed with RIPA buffer and proteinase inhibitor mixture (PMSF). Nuclear and cytoplasm protein were separated by using nuclear and cytoplasm protein extraction kit (78833; Thermo Pierce, Rockford, IL, USA), according to manufacturer's instructions. Protein concentration was assessed by Bradford assay (Bio-Rad Laboratories, Shanghai, China). Total proteins were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PerkinElmer, Boston, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 hr at room temperature and incubated overnight with primary anti-col3A1, anti-col1A1, anti-β-actin (sc-28888, sc-8784, sc-47778, respectively, Santa Cruz biotechnology Inc, Santa Cruz, CA, USA), anti-OPN (ab8448; Abcam) at 4°C, respectively. After washing, HRP-conjugated secondary antibody was added for 1 hr at 37°C. Detection was performed with enhanced chemiluminescence (ECL) and relevant blots were quantified by densitometry by using the accompanying computerized image analysis program.

Withaferin A was procured from Aptus laboratories (Hyderabad, India) and TGF-β1 was obtained from Bio-legend (United States); Bleomycin sulfate was procured from Cipla labs (India); Masson’s trichrome staining kit, Sirius red, Chloramine-T, Hydroxyproline, and Ehrlich reagent were procured from Sigma-Aldrich, Anti-ZO-1, anti-E-cadherin, anti-Smad 2/3, anti-p Smad 2/3, anti-vimentin, anti-NF-κB p65, anti-p VEGF, anti-p p38 MAPK, anti-p FAK, and anti-p PLCγ1 were procured from Cell Signaling Technology, while anti-Col 1A2, anti-Col 3A1, anti-smooth muscle actin, anti-CTGF, anti-fibronectin, and anti-TGF-β1 were obtained from Santa Cruz Biotechnology (United States). ELISA kits were purchased from eBioscience, United States. TGF-β bioplex kit was procured from Merck-Millipore. Rest of the chemicals and reagents were of analytical grade and obtained from commercially available sources.