Chemidoc imaging system version 2

One section of the heart was lysed in the complete RIPA lysis buffer and stored at −80 ◦C for immunoblotting analysis. Immunoblotting analyses were performed according to what has been previously published (Reis-Mendes et al. 2021a (link), b (link)). The enhanced chemiluminescence ECL reagents were used to detect immunoreactive bands, according to the manufacturer’s instructions. Digital images were acquired using the ChemiDoc Imaging System version 2.3.0.07 (Bio-Rad, Hercules, CA, USA). The images obtained were analyzed using the Image Lab software version 6.0.1 (Bio-Rad, Hercules, CA, USA). Protein content in the whole cardiac homogenate was quantified using the Bio-Rad DC Protein assay. Protein loading of Western blotting was confirmed by Ponceau S staining, as done by us in other works (Reis-Mendes et al. 2021a (link), b (link)). 

Heart sections were lysed in RIPA buffer through sonication and were kept at −80 °C until analysis. Samples containing 20 µg of protein were then processed, as previously described [27 (link)]. The slot-blot membranes were blocked with 5% (w/v) dry non-fat milk in TBS-T (100 mM Tris, 1.5 mM NaCl, pH 8.0 and 0.5% Tween 20) for 1 h and then incubated for 1 h with primary antibody (rabbit anti-DNP, 1:1000) in 5% (w/v) non-fat dry milk in TBS-T. The membranes were washed three times (10 min each) with TBS-T and incubated for 1 h with a solution of anti-rabbit IgG-peroxidase (1:5000). Immunoreactive bands were detected using the ECL reagents, according to the supplier’s instructions, and digital images were acquired using the ChemiDoc Imaging System version 2.3.0.07 (Bio-Rad, Hercules, CA, USA). The images obtained were analyzed using Image Lab software version 6.0.1 (Bio-Rad, Hercules, CA, USA).

Heart sections were lysed in RIPA buffer (supplemented with PMSF, DTT, NaF, Na₃VO_4, and a cocktail of protease inhibitors) through sonication and were kept at −80 °C until analysis. Samples containing 20 µg of protein (as assessed by the Bio-Rad DC Protein assay) were then processed as previously described [75 (link)]. Immunoreactive bands were detected, and digital images were acquired using the ChemiDoc Imaging System version 2.3.0.07 (Bio-Rad, Hercules, CA, USA). The obtained images were analysed with Image Lab software version 6.0.1 (Bio-Rad, Hercules, CA, USA).