Sarcomeric alpha actinin

Cell or myobundle protein was isolated in RIPA lysis and extraction buffer with protease inhibitor (Sigma). Protein concentration was determined using BCA assay (Pierce of Thermo Scientific, Rockford, IL) according to manufacturer's instructions. Western blot was performed using Bio-Rad Mini-PROTEAN gels and the Mini-PROTEAN Tetra cell, Mini Trans-blot module (Bio-Rad, Hercules, CA). The following primary antibodies were used for detection: GAPDH (SCBT, 1:500), LC3 (Cell Signaling, 1:200), muscle creatine kinase (SCBT, 1:200), myosin heavy chain 1/2/4/6 (SCBT, 1:200), and sarcomeric alpha-actinin (Sigma, 1:200). HRP conjugated anti-mouse (1:20,000) and anti-goat (1:5000) antibody were purchased from Sigma, and HRP conjugated anti-rabbit was purchased from SCBT (1:5000). Chemiluminescence was performed using Clarity Western ECL substrate (Bio-Rad). Images were acquired using a Bio-Rad Chemidoc and analyzed using ImageJ.

For the detection of protein phosphatases and endogenous protein phosphatase inhibitors, cells isolated from rabbit LV and sinoatrial nodes were lysed, transferred to membranes, incubated with antibodies against PPP1CA (Sigma-Aldrich, St. Louis, MO, USA SAB5300221, 1:2500), PPP2CA (Millipore, Bedford, MA, USA 06-222, 1:1000), Inhibitor 1 (Abcam, Cambridge, MA, USA ab40877, 1:10000), KEPI (Biorbyt, Cambridge, UK orb128558, 1:2000), DARPP-32 (Santa Cruz Biotechnology, Dallas, TX, USA sc-271111, 1:1000), or Sarcomeric Alpha-Actinin (Sigma-Aldrich, St. Louis, MO, USA A7811, 1:5000) and developed with Pierce SuperSignal West Pico or West Dura ECL substrate kits (ThermoFisher Scientific, Waltham, MA, USA).
The detection of site specific PLB phosphorylation sites was performed in SANC as previously described [24 (link)].