Collagen 1a1

Methods for immunohistochemical staining are described elsewhere 26 (link). Antibodies used for IHC were fibronectin (Abcam), collagen1A1 (Santa Cruz), TGF-β (Abcam), and IL-1β (Santa Cruz). Primary antibody labeling detection was performed after incubation with the appropriate DAB incubated secondary antibody. For fluorescent labeling of Nrf2 nucleus translocation, frozen sections were fixed with 4% PFA for 10 min and permeabilized with 0.1% Triton X-100 for 20 min. After blocking with 5% BSA for 1h, sections were stained with Nrf2 antibody (Abcam), and then stained with secondary antibody, Texas Red (Invitrogen). Nuclei were counterstained with hematoxylin in IHC experiments and with DAPI in SlowFade® Gold Anti-fade Mountant in IF experiments.

Tissue lysates were prepared according to standard protocols with slight modifications^{15 (link)}. In brief, 1 mL of RIPA buffer containing 0.1% sodiumdodecylsuphate (SDS) with phosphatase and protease inhibitors (Roche Diagnostics) was added to approximately 50 mg of heart tissue. The tissue was further homogenized and protein concentration was determined using DC assay (Bio-Rad). Tissue lysates containing 30 μg protein/well were further subjected to SDS-PAGE separation and proteins were transferred to PVDF membranes. Next, membranes were blocked with 5% skimmed milk. Further, membranes were cut at 55 kDa and primary antibodies was applied separately on each section of the membrane (upper part: Collagen 1a1, Santa Cruz, sc-293182, dilution 1:500; lower part: β-actin, Thermo Fischer Scientific, PA1-183, dilution 1:10 000), and incubated at 4 °C for 16 h. After a series of washes in TBS-T, a corresponding HRP-labeled secondary antibody (Dianova, dilution 1:10 000) was next incubated for 1 h at room temperature. Chemoluminescence was detected by Pierce ECL Substrate and using a ChemiDoc Imaging system (BioRad). Ponceau staining (BioRad) was used to visualize protein transfer. Relative protein levels were determined by ImageJ (NIH, version 1.8.0_66).

Western blot analysis of fibroblast lysates and supernatants was done as previously described [11 (link)]. The following antibodies were used: Anti-COL22A1 (Novus, Littleton, CO, USA), fibronectin, collagen 1A1, CTGF, GAPDH (Santa Cruz, Dallas, TX, USA), ACTA2 (Sigma-Aldrich), and horseradish peroxidase-labeled secondary antibody (Santa Cruz). Signals were detected by chemiluminescence (ProteinSimple, San Jose, CA, USA).