sHA 14-1 inhibits SERCA enzymatic activity. NALM-6 (A) and JURKAT (B) cells were preloaded with Fura-2AM for 30 min. The cells were then incubated with 100 nM thapsigargin (+), the minimal concentration to induce full Ca2+ release, and increasing concentrations of sHA 14-1, as shown in the grid below the figures. Calcium release was quantified using fluorescent changes at excitation wavelengths of 340 and 380 nm monitored on a dual-wavelength fluorometer. Data are mean ± S.D. (n = 3 for each conditions). *, p < 0.05; **, p < 0.01; ***, p < 0.0001 compared with the quantity of calcium released by TG alone. C, increasing concentrations of sHA 14-1 were incubated for 10 min at 37°C with SR membrane fractions purified from rabbit skeletal muscle. Rabbit skeletal muscle Ca2+ ATPase activity (corresponding to SERCA 1A) was measured using the phosphate liberation assay, as described previously (Wootton and Michelangeli, 2006). Data are mean ± S.D. (n = 3 for each conditions). D, increasing concentrations of sHA 14-1 were incubated for 10 min at 37°C with pig brain microsomes. Ca2+ ATPase activity in these membranes (corresponding to SERCA 2B) was measured using a phosphate liberation assay, as described previously. Data are mean ± S.D. (n = 3 for each conditions). E, a SERCA sample (99%, 20 μg) was analyzed for the presence of Bcl-2 protein by Western blot with NALM-6 protein lysate (0.2, 1, and 5 μg) as a positive control. The results are representative of three separate experiments.