Calcineurin

Total RNA of homogenized murine whole heart or cultured cardiomyocytes was isolated using Total RNA Kit II (Omega) according to the protocol provided by the manufacturer. Conventional or quantitative real-time PCR using a Quantitect SYBR Green RT-PCR kit (QIAGEN) and an Applied Biosystems 7500 system targeting the genes of Ankrd1, ANP, β-MHC, calpain 1, early growth response 1 (egr-1), β-actin and GAPDH, was performed. Primers were showed in Table 1 and 2.
Proteins were extracted from the cultured cardiomyocytes or their mitochondria, or the murine heart. Immunoblotting was performed by using antibodies against CARP (Santa Cruz), calcineurin (Abcam). Blotting of β-actin or GAPDH (Santa Cruz) was used as a loading control.

Control and FGF23 (1 ng/mL)-treated PV cardiomyocytes were centrifuged and washed with cold PBS, and lysed on ice for 30 min in RIPA buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), and protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration were determined with a Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Proteins were separated in 4%~12% SDS- polyacrylamide gel electrophoresis (PAGE) under reducing conditions and electrophoretically transferred into an equilibrated polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, UK). All blots were probed with primary antibodies against SERCA2a (Santa Cruz Biotechnology, Santa Cruz, CA, USA), pCaMKII at Thr 286, calcineurin (Abcam, Cambridge, United Kingdom), CaMKII, PLB pSer16 (GeneTex, San Antonio, TX, USA), RyR pS2808, RyR pS2814, PLB pThr17 (Badrilla, Leeds, UK), RyR channels, total PLB (Thermo, Rockford, IL, USA), α-actin (Sigma, St. Louis, MO, USA), and all secondary antibodies conjugated with horseradish peroxidase. All bound antibodies were detected with an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA) and analyzed with AlphaEaseFC software. All targeted bands were normalized to α-actin to confirm equal protein loading.