Ma3 919

Total RNA was extracted using TRIzol (Invitrogen) following the provider's recommendations and retrotranscribed into cDNA using iScript Supermix (Bio-Rad) following the manufacturer's instructions. Real-time qPCR was performed using Taq-man probes (Applied Biosystems) for Tnnt2, Myh6, and Actc1, and Kapa Sybr Fast (Kapa Biosystems) for IL33 using the primers Fw 5′-TCC AAC TCC AAG ATT TCC CCG-3′ and Rv 5′-CAT GCA GTA GAC ATG GCA GAA-3′. For input normalization, we used Gapdh Fw 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and Rv 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′. Western blot analyses were performed as described by Nelson et al. (2016) (link), using SERCA2 ATPase antibody (1:1,000, MA3-919, Thermo Scientific), ryanodine receptor antibody (1:1,000, MA3-916, Thermo Scientific), and MEF2C antibody (1:500, ab79436, Abcam). ELISA was performed using Mouse IL33 DuoSet from R&D.

The following primary antibodies were used: mouse monoclonal antibodies to α-actinin [Western blotting (WB), 1:2500; immunofluorescence (IF), 1:400; A7811, Sigma-Aldrich], actin (WB, 300 ng/ml; A2171, Sigma-Aldrich), myomesin [WB/IF, 1:10; mMaC, Developmental Studies Hybridoma Bank (DSHB)], myosin (WB/IF, 1:10; MF20, DSHB), RyR2 (WB/IF, 1:1000; MA3-925, Thermo Fisher Scientific), SERCA2 (WB, 1:1000; IF, 1:100; MA3-919, Thermo Fisher Scientific), PLN [WB, 1:5000 (A010-14; Badrilla); WB, 2 μg/ml; IF, 1:200 (ab2865, Abcam)], titin (IF, 1:10; 9D10, DSHB), obscurin-NH₂ (WB/IF, 1:10; tissue culture supernatant) (13 (link)), and Hax-1 (WB, 1:1000; clone 52; 610824, BD Biosciences); rabbit monoclonal antibody to HSP90 (WB, 1:1000; 4877, Cell Signaling); and rabbit polyclonal antibodies to dihydropyridine receptor (WB/IF, 1:200; ab58552, Abcam), junctophilin-2 (WB, 1 μg/ml; 40-5300, Invitrogen), titin-Z (IF, 2 μg/ml) (44 (link)), SERCA2 (IF, 1:100; Ab91032, Abcam), sAnk1.5 (WB, 300 ng/ml; IF, 3 μg/ml) (9 (link)), SERCA2-pSer38 (WB, 1:1000; IF, 100; A010-25AP, Badrilla), RyR2-pSer2808 (WB, 1:2000; IF, 1:100; ab59225, Abcam), RyR2-pSer2814 (WB, 1:1000; A010-31AP, Badrilla), PLN-pSer16 (WB, 1:5000; A010-12, Badrilla), PLN-pSer16 (IF, 1:200; 07-052, Millipore), obscurin-COOH (WB, 300 ng/ml) (9 (link)), and obscurin-Ig58/59 (IF, 3 μg/ml) (45 (link)).

Protein extracts (20 μg/sample) from LV tissues were analyzed by SDS‐PAGE and subjected to western blotting analyses using specific antibodies and the enhanced chemiluminescence method (SuperSignal West Pico chemiluminescent substrate; Pierce, Rockford, IL). The primary and secondary antibodies used in our experiments were the following: mouse anti‐Na⁺/Ca²⁺ exchanger (NCX) and anti‐sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA‐2A; MA3‐926 and MA3‐919, respectively; Thermo Fisher Scientific, Waltham, MA); goat antiphosphorylated phospholamban (p‐PLN; sc‐12963; Santa Cruz Biotechnology, Dallas, TX); rabbit anticalsequestrin (ab62662; Abcam, Cambridge, MA); and rabbit anti‐troponin‐I (TnI; 4004; Cell Signaling Technology, Danvers, MA). Films were scanned and analyzed using Image Lab software (version 4.1; Bio‐Rad). Band density of the protein of interest was normalized to beta‐actin (A1978; Sigma‐Aldrich, St. Louis, MO) or extracellular signal‐regulated kinase 1 and 2 (ERK 1&2; sc‐93 and sc‐154; Santa Cruz Biotechnology).