Myofibril pellets were washed twice with reaction buffer (25 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg/mL BSA). Washed myofibril lysates were resuspended in 200 μL reaction buffer with or without recombinant HDAC6 (1 μg/μL; BPS Bioscience) for 30 minutes at 15°C. For the treatment of recombinant PKCα and recombinant HDAC6, myofibril lysates were resuspended in 150 μL of relaxing solution containing recombinant PKCα (0.066 U/μL; Millipore 14-484) and lipid activator (Millipore 20-133) for 30 minutes with or without the addition of recombinant HDAC6 (1 μg/μL; BPS Bioscience) for another 30 minutes. After treatment, myofibril lysates were washed twice with relaxing solution containing protease inhibitors, and mechanics studies were performed. Homogenized myofibrils were centrifuged at 13,000g for 5 minutes, then separated using agarose gels.
Lin Y.H., Major J.L., Liebner T., Hourani Z., Travers J.G., Wennersten S.A., Haefner K.R., Cavasin M.A., Wilson C.E., Jeong M.Y., Han Y., Gotthardt M., Ferguson S.K., Ambardekar A.V., Lam M.P., Choudhary C., Granzier H.L., Woulfe K.C, & McKinsey T.A. (2022). HDAC6 modulates myofibril stiffness and diastolic function of the heart. The Journal of Clinical Investigation, 132(10), e148333.
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