Anti serca2

Cells and tissues were homogenized in ice-cold RIPA (50 mM Tris pH 8.0, 1% nonidet P40 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl or ThermoFisher, 89900) or urea containing phosphatase inhibitor cocktail 3 (Sigma-Aldrich) and protease inhibitor (Roche Diagnostics or ThermoFisher, 78440) and 15 mM NaVanadate in selected experiments. Protein concentrations were determined with a DC protein assay kit (BioRad) or BCA protein Assay Kit (Pierce, 23225). Equal amounts of protein were loaded on 10% polyacrylamide gels or a Mini Protein precast gel (Biorad, 4561085). After electrophoresis, the gels were blotted onto PVDF or nitrocellulose membranes. Membranes were then blocked and incubated overnight at 4 °C with the primary antibody, followed by 1 h incubation at room temperature with an HRP secondary antibody. Detection was performed by ECL and analyzed using ImageJ version 2.0.0. Protein quantifications were normalized to total protein staining (Revert 700) or GAPDH protein expression levels. Primary antibodies used: anti-PLN monoclonal (clone 2D12) (ThermoFisher: #MA3-922 1:1000), anti-SERCA2 (ThermoFisher: #MA3-919 1:1000 or Abcam ab150435 1:1000–1:5000), phospho-PLN (Cell Signaling: #8496 1:1000), GAPDH-HRP (1:2000, 1:35.000, Invitrogen MAS-15738).

About 1 g of left ventricle wall close to the apex of heart was cut on ice, and homogenized in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL, USA). 40 μg/sample proteins were resolved with 10 % SDS-PAGE and transferred to membrane. Immuno-staining was performed by standard techniques with primary antibodies as follows: anti-GAPDH from Cell Signaling Technology (Boston, MA, USA); anti-ACTA1 from ABGENT (San Diego, CA, USA); anti-SERCA2 from Thermo Fisher Scientific (Rockford, IL, USA); and anti-MYH7 from GeneTex (Irvine, CA, USA). Signals were detected by supersignal™ chemiluminescence (Pierce, Rockford, IL, USA) and analysed by densitometry.

Cells fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 were incubated with anti-cTnT (1:100, Abcam, Cambridge, UK), anti-α-MHC (1:100, Abcam, Cambridge, UK)), anti-α-SA (1:100, Abcam, Cambridge, UK), anti-CACNA1C (1:100, Abcam, Cambridge, UK), and anti-SERCA2 (1:100; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), followed by the appropriate secondary antibody conjugated with Alexa Fluor 488 or PE (1:200; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), as previously reported [51 (link)]. Nuclei were counterstained with DAPI (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Samples treated only with the secondary antibody were used as controls. Images were acquired using an Axio Vert A1 microscope. Images were analyzed by ZEN 2009 (Carl Zeiss, Oberkochen, Germany).