P camkii

Protein expression analysis was performed according to manufacturer’s instructions (Bio-Rad). In brief, proteins were subjected to SDS-PAGE (4–20% acrylamide separating gel, Criterion stain-free TGX Biorad). The separated proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA), which was incubated overnight with the diluted antibody. Primary antibodies against CREB, pCREB, pERK, Sirt1, and c-Fos, and secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA); CAMKII, pCAMKII, ERK, and PKC were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Immunoblots were visualized by means of a chemiluminescence reaction (Millipore) by Image LabTM Software (Biorad) under a luminescent image analyzer (Chemidoc XSR+ Bio-Rad). Only bands below the saturation limit were analyzed. The protein level was normalized to the optical density of total proteins in each lane.

Slices used for coimmunoprecipitation analyses were preincubated with Tat-GluN2B (ChinaPeptides). Total protein was incubated with antibody against CaMKII (2 μg) overnight at 4°C in immunoprecipitation buffer (0.05 M HEPES, pH 7.4, containing 10% glycerol, 0.15 M NaCl, 1% TritonX-100, 0.5% Nonidet P-40, and 1 mM concentration of each of EDTA, EGTA, PMSF, and Na₃VO₄). Then protein A/G-agarose beads were added, mildly vortexed, and incubated for 2 h at 4°C. The beads were recovered by centrifugation at 12,000 ×g and gently washed three times with immunoprecipitation buffer. The left beads were treated the same as Western blot. For Western blot, total protein (20 μg) was boiled and was separated with 7.5% SDS-PAGE and transferred onto a PVDF membrane. Membranes were blocked in 3% (w/v) BSA (fraction V) in TBST (0.1% Tween 20) for 1 hr at room temperature (RT). Detection antibodies for Western blot analysis were from Biotime (β-Tubulin, 1 : 3000), Santa Cruz (p-CaMKII, 1 : 800), and Santa Cruz (GluN1, 1 : 800). Conjugated antibodies were from GE Healthcare (anti-mouse IgG-HRP, 1 : 4000) and Santa Cruz (rabbit anti-goat IgG-HRP, 1 : 8000). Signals were visualized with ECL kit (Amersham Biosciences). Band intensities were quantified by ImageJ (NIH, USA).

Western blot analysis was performed by a standard method on proteins isolated from monolayers from control- and DMD-hiPSC-cardiomyocytes, as previously described (Ferrantini et al., 2017 (link)). The following antibodies were used: p-RYR2 S2808 (Badrilla); p-RYR2 S2814 (Badrilla); p-CAMKII (Santa Cruz); CAMKIIδ (Abcam); GAPDH (Millipore). Relative intensity of individual bands from Western blots was quantitated using ImageJ software and normalized to GAPDH or total CaMKII (Supplementary Figure S7).