Ecl peroxidase labeled anti mouse antibody

Proteins were extracted using RIPA Buffer (Wako) according to the manufacturer’s recommendation, and protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were run by SDS–PAGE using Criterion TGX Precast Gel (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). The primary antibodies used were anti-ERRγ polyclonal antibody (Abcam, ab49129, 1:2000 dilution), anti-cardiac Troponin I (cTnI) (Thermo, MA1-20112, 1:250 dilutiion), anti-Troponin I1 (ssTnI) (abcam, EPR17120-11, 1:1000 dilution), anti-GAPDH (Cell Signaling, 5174, 1:1000 dilution) and anti-actin monoclonal antibody (Merck Millipore, MAB1501, 1:5000 dilution). The secondary antibodies used were ECL peroxidase-labeled anti-mouse antibody (GE Healthcare, NA931, 1:5000 dilution) and ECL peroxidase-labeled anti-rabbit antibody (GE Healthcare, NA934, 1:5000 dilution).

Western blot was conducted as previously described^{37 (link)}. Purified schizont-rich parasite pellets from P. falciparum 3D7 strain were directly lysed in reducing or non-reducing SDS-PAGE sample buffer. The lysate was boiled at 95 °C for 5 min, centrifuged at 10,000 × g for 10 min at 4 °C, supernatant collected, and resolved by SDS-PAGE on a 12.5% e-PAGEL (ATTO). Following electroblotting, a polyvinylidene fluoride membrane was incubated with primary antibodies diluted at the following concentrations: rabbit anti-PfPV1 antibody, 1:1000; mouse anti-PTP5 antibody, 1:1000; rat anti-HA monoclonal antibody (Roche Diagnostics, Indianapolis, IN, USA), 1:100; mouse anti-PF3D7_0801000 antibody, 1:1000. After washing, the membrane was incubated with secondary antibodies diluted at the following concentrations: ECL peroxidase labeled anti-mouse antibody (GE Healthcare), 1:10000; ECL peroxidase labeled anti-rabbit antibody (GE Healthcare), 1:10,000; ECL peroxidase labeled anti-rat antibody (GE Healthcare), 1:1000.

Cultured murine cells were lysed in RIPA buffer (Nacalai Tesque, Kyoto, Japan). Protein concentration was determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). The denatured lysate mixed with Novex™ Tris–Glycine SDS Sample Buffer (2×; Thermo Fisher Scientific) was loaded onto Novex™ WedgeWell™ 4–12% Tris–Glycine gels (Thermo Fisher Scientific) for electrophoresis. The gels were electroblotted onto a PVDF membrane using iBlot 2 PVDF mini stacks (Thermo Fisher Scientific). Membranes were blocked with Blocking One (Nacalai Tesque) for Nfatc1 detection and with 5% skim milk (FUJIFILM Wako Pure Chemical Corporation) for β-actin. The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies at room temperature for 1 h. ECL Prime Western Blotting Detection Reagent (GE Healthcare Bioscience, Chicago, IL, USA) was added to the membranes, and the chemiluminescent signal was detected using a CCD camera (Vilber, Collégien, France). The primary antibodies used were mouse monoclonal antibodies against Nfatc1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7294, 1:1000 dilution) and β-actin (Sigma-Aldrich, A1978, 1:2000 dilution). The secondary antibody was an ECL peroxidase-labeled anti-mouse antibody (GE Healthcare Bioscience. 1:10,000 dilution).