Ponceau staining

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).