Ab10231

Whole tissue lysates were prepared to determine troponin protein levels. Therefore, pulverized frozen tissue was homogenized in 40 μl/mg tissue 1× reducing sample buffer (106 mM Tris-HCl, 141 mM Tris-base, 2% lithium dodecyl sulfate (LDS), 10% glycerol, 0.51 mM EDTA, 0.22 mM SERVA Blue G250, 0.18 mM Phenol Red, 100 mM DTT) using a glass tissue grinder. Proteins were denatured by heating to 99 °C for 5 min and debris was removed by centrifugation at maximum speed for 10 min in a microcentrifuge (Sigma, 1–15 K).
For analysis of troponin protein levels by Western blot, 5 μg of protein were separated on a 4–15% TGX gradient-gel (Biorad) and transferred to a polyvinylidene difluoride membrane. Site-specific antibodies directed to cTnT (ab10214, Abcam), cTnT (T6277, Sigma-Aldrich), cTnT (ab8295, Abcam), cTnI (ab10231, Abcam), cardiac Troponin C (cTnC, sc48347, Santa Cruz) and α-actinin (A7811, Sigma-Aldrich) were used to detect the proteins which were visualized with an enhanced chemiluminescence detection kit (Amersham) and scanned with Amersham Imager 600. Protein levels were determined by densitometric analysis. Protein levels were normalized to α-actinin or cTnI when appropriate.
Equal loading of troponin complexes was verified with Imperial protein stain (Thermo Scientific).

The troponin complex was exchanged in membrane‐permeabilized cardiomyocytes as previously described (Wijnker et al. 2013). The recombinant WT or TNNT2_p.K217del troponin complex was added to the cells in a concentration of 1 mg ml⁻¹. The recombinant troponin complexes could be distinguished from highly phosphorylated endogenous troponin since the recombinant troponins were not phosphorylated. Quantification of the exchange rate was performed by phos‐tag analysis in which non‐, mono‐ and bis‐phosphorylated cTnI (Pierce, Rockford IL, USA, MA1‐22700) were separated by polyacrylamide‐bound Mn²⁺‐phos‐tag gel electrophoresis Western blotting as previously described (Najafi et al. 2015). The percentage of recombinant troponin complex present after exchange was quantified as the percentage of non‐phosphorylated cTnI to the total of non‐, mono‐ and bis‐phosphorylated cTnI levels. Total cTnI levels after exchange were quantified by cTnI (Abcam, ab10231) and corrected for myosin light chain‐2 (MLC2) (Enzo, Farmingdale, NY, USA, ALX‐BC‐1150).