YM-58483 (BTP2) is a specific and effective inhibitor of CRAC channels and subsequent Ca2+ signals.

HCT-116 cells are washed with PBS and then homogenized with a 27-gauge syringe in binding buffer (10 mm Tris-HCl (pH 7.4), 50 mm KCl, 5 mm MgCl2, 1 mm EDTA, and 0.1 mm Na3VO4). The cell lysate is centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant is collected. The HCT-116 cell lysate supernatant is precleared by incubating with Dynabeads M-280 streptavidin for 30 min at 4°C and captured by magnet separation. The cleared supernatants are incubated with biotinyl-KRIBB11 compound. After overnight incubation at 4°C, proteins associated with the biotinyl-KRIBB11 compound are precipitated with Dynabeads M-280 streptavidin. Precipitated samples are separated by a magnet. Samples are washed with 1 mL of ishing buffer containing 50 mm HEPES (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.1% Tween 20, 10% (v/v) glycerol, 1 mm NaF, 0.1 mm Na3VO4, and protease inhibitor mixture tablets (1 tablet/10 mL). Samples are boiled in SDS-PAGE sample buffer, separated by 10% polyacrylamide gel, and immunoblotted with antibodies against HSF1, HSF2, HSP90, or CDK9.

Jurkat cells (1×107 cells/ml) were tested with varying concentration of compounds for 30 min at 37°C. The cells were stimulated with 1 μM ionomycin for 30 min at 37°C. After stimulation, the cells were centrifuged at 200×g for 2 min, and were solubilized in 100 μl of Triton X-100 lysis buffer. The cell lysate was centrifuged at 15,000×g for 20 min; the clarified lysate was subjected to SDS-PAGE; and NF-ATc2 was detected by Western blotting with anti-NF-ATc2 mAb. (Only for Reference)

YM 58483, BTP2

Ethanol: 78 mg/mL (185.13 mM), Sonication is recommended.

H2O: < 1 mg/mL (insoluble or slightly soluble)

DMSO: 250 mg/mL (593.37 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (4.75 mM), Sonication is recommended.