Protease inhibitor cocktail

For the stage-specific pull-down analysis parasites were harvested at ring stage (approx. 12HPI), trophozoites (approx. 24HPI) and schizonts (approx. 36HPI) Parasites were liberated, and red blood cells lysed using 10 volumes of 0.1% (w/v) saponin (Sigma) in ice-cold 1 x PBS (1^st Base) for 5 minutes. Parasite pellet was washed three times with 1 x PBS and the pellet was lysed (for most of assays except Pull-downs) with RIPA buffer (150 mM sodium chloride (Merck); 1% Triton X-100 (BioRad); 0.5% sodium deoxycholate (Sigma); 0.1% sodium dodecyl sulfate (BioRad); and 50mM Tris (BioRad) pH8.0) supplemented with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque) and 1% (v/v) phosphatase inhibitor cocktail (Thermo Fisher). Samples were incubated for 30 min at 4°C with gentle agitation. For native pull-down assays RIPA buffer was substituted with Pierce IP Lysis Buffer (Thermo Fisher) with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque) and 1% (v/v) phosphatase inhibitor cocktail (Thermo Fisher). Protein lysate debris was removed by centrifugation (15 min at 4°C) and protein concentration in the supernatant was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were frozen with liquid nitrogen and stored at -80°C.

For co-immunoprecipitation, 6-well plate HEK293T cells were transfected by the calcium phosphate method. Two days after transfection, cells were lysed in NP-40 lysis buffer (20 mM Tris-HCl pH 7.5, 250 mM NaCl, 1% NP-40) supplemented with 1 mM PMSF, protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitor cocktail (Roche). Total cell lysates were precleared on Protein A Sepharose beads for 30 min at 4°C. The precleared cell lysates were immunoprecipitated with Protein A beads-conjugated with the desired antibodies for 6 hr. Immunoprecipitates were washed three times with lysis buffer.
To detect the protein interaction in JPM50.6/WT cells, 1×10⁸ cells were lysed in NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitor cocktail (Roche). Total cell lysates were precleared on Protein A Sepharose beads for 30 min at 4°C. The precleared cell lysates were immunoprecipitated with Protein A beads-conjugated with 2 µg of anti-CKIP-1 Ab at 4°C overnight. Immunoprecipitates were washed three times with 0.05% NP-40 buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40).

Western blotting was performed as described previously^{14 (link)}. The isolated LV tissues of non-infarcted were homogenized with Cell destroyer (Bio Medical Science Inc., Tokyo, Japan), and total protein of the tissues was extracted by RIPA lysis buffer (20 mM Tris–HCl pH 7.4, NaCl 150 mM, MgCl₂ 10 mM, 1% TritonX-100, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate) containing 1% protease inhibitor cocktail (Nacalai Tesque). Total protein of cardiac fibroblasts was extracted by cell lysis buffer (Cell signaling Technology) containing 1% protease inhibitor cocktail (Nacalai Tesque). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to separate equal amounts of proteins (LV tissue lysate: 20 μg, cell lysate: 1 or 10 μg), and the proteins were transferred to a nitrocellulose membrane (Pall Corporation, Ann Arbor, MI, U.S.A.). The membranes were incubated in 0.5% skim milk for blocking non-specific binding of antibody to antigen. After overnight incubation with the primary antibody at 4 °C, the membranes were incubated with secondary antibody. The chemiluminescent signal was detected by EZ-ECL detecting reagents (Biological Industries, Kibbutz Beit, Haemek, Israel) using an ATTO light capture system (AE-6972; ATTO, Tokyo, Japan). The images of chemiluminescent signals were analyzed with a CS Analyzer 3.0 software (ATTO).

Tissues were homogenized using a Polytron homogenizer (PT 2100, Brinkman Instruments, Inc., Westbury, NY) in 2 mL of PBS for measurement of cytokines and Cu ions, and in a lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA) containing protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan) for Western blotting and for measurement of ROS levels and SOD activity. Cells were homogenized in a protease inhibitor cocktail (Nakalai Tesque)-containing lysis buffer and subjected to Western blotting and measurement of ROS levels and SOD activity. The homogenates were clarified by centrifugation at 1,000×g for 2 min at 4°C. Protein concentration of the homogenates was measured using the BCA method (Thermo Scientific, Rockford, IL).