Ecl select chemiluminescence kit

NRVCMs were harvested in lysis buffer and lysed by three freeze and thaw cycles. For protein isolation from murine hearts, tissue was shredded in lysis buffer. To remove cell debris a centrifugation step (12,000 g, 20 min, 4°C) was carried out. Protein concentration was determined photometrically on an Infinite m200 PRO system (Tecan) by using the Bradford protein assay kit according to the manufacturer’s manual (Bio-Rad). Protein samples were resolved by 10% SDS-PAGE and transferred onto a polyvinylidenefluoride (PVDF) membrane. After blocking in 5% dry-milk in Tris-buffered saline with Tween20 (TBS-T), the incubation with the target-specific primary antibody was carried out overnight at 4°C, followed by application of a suitable horseradish peroxidase-coupled secondary antibody (Supplementary Table S1). For visualization of protein bands, an ECL Select chemiluminescence kit (GE Healthcare) and the Flourchem Q imaging system (Biozym) was used. Quantitative densitometric analysis was carried out with ImageJ/Fiji version 1.46. Tubulin, GAPDH or whole protein content stained by Ponceau was used for normalization.

Cells and tissues were homogenized using RIPA buffer (MilliporeSigma) supplemented with protease and phosphatase inhibitors (Pierce, Thermo Fisher Scientific). Total protein (10 μg) was heated for 10 minutes at 70°C and loaded onto NuPAGE Novex gels (Thermo Fisher Scientific). After transferring the proteins onto nitrocellulose membranes, the membranes were blocked with 5% milk in TBS-Tween followed by incubation with anti–cleaved caspase 3 (1:1,000; no. 9661, Cell Signaling Technology); anti–cleaved PARP1 (1:1,000; no. 5625, Cell Signaling Technology); anti–cyclin D1 (1:1,000; sc-718, Santa Cruz Biotechnology); or anti-PCNA (1:1,000; no. 13110, Cell Signaling Technology) primary antibodies. Visualization of proteins was achieved using HRP-coupled secondary antibodies (1:2,000; no. 7074, Cell Signaling Technology). Signal detection was performed using the ECL Select Chemiluminescence Kit and ImageQuant LAS 4000 (both from GE Healthcare). Data were analyzed with ImageJ. Membranes were probed with anti-GAPDH (G9545, MilliporeSigma) or anti–β-actin (4970, Cell Signaling Technology) antibodies as normalizers.

NRVCMs were lysed 72 h post viral transduction by three freeze–thaw cycles in RIPA buffer (20 mM Tris, 10 mM DTT, 500 mM sodium chloride, 1% NP40, 12,5% glycerol) supplemented with phosphatase and protease inhibitor cocktails (Roche, Germany). The cell lysate was then centrifuged at 12,000 × g for 20 min. to remove cell debris, and the supernatant was used for protein concentration measurement by DC-assay (Bio-Rad Laboratories). Proteins prepared as above were resolved on 10% SDS–PAGE, or commercially available 4–12% gradient gels (Life Technologies), and transferred to a polyvinylidenefluoride membrane, blocked for 2 h in 5% dry-milk prepared in 0.1% TBST at room temperature (RT), followed by the incubation with primary antibodies overnight at 4 °C, 4× washes with 0.1% TBST and final incubation with a suitable HRP-coupled secondary antibody (1:10,000) (Santa Cruz, Germany). Protein signals were developed using ECL-select chemiluminescence kit (GE Healthcare) and visualized on Fluorchem Q imaging system (Biozym). Quantitative densitometry was performed with the help of ImageJ/Fiji version 1.46. All the uncropped immunoblot images have been included as Supplementary Fig. 7. All source data underlying the graphs presented in the main figures using immunoblot densitometry data are made available as Supplementary Data 1.