FIGURE 4.

Effect of an increase in intracellular free calcium on the association of eNOS and calpain with HSP90. bEnd5 cells (1 × 10⁶ cells) left untreated (Control) or treated with 1 μm Ca²⁺-ionophore A23187 for 5 min at 37 °C in the absence (+Ca²⁺) or in the presence of 2 mm Mg-ATP, together with 10 mm phosphoenolpyruvate and 2 μg of purified pyruvate kinase (+Ca²⁺+ATP) were lysed, and aliquots (500 μg of soluble protein) obtained as described under “Experimental Procedures” were incubated overnight at 4 °C with monoclonal anti-HSP90 antibody. The mixtures were then incubated for 1 h at 4 °C with protein G-Sepharose (30 μl). The particles were collected, washed three times with the immunoprecipitation buffer, resuspended in SDS/PAGE loading solution (30 μl), and submitted to 6% SDS/PAGE. The presence in the solubilized material of eNOS and calpain together with HSP90 was established using the specific mAbs. The immunoreactive bands of each protein were quantified as described under “Experimental Procedures.” The values of each quantification are the arithmetical mean ± S.D. of three different experiments.