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| Figure 1: Effect of external Ca2++, EGTA or LaCl3 on free intracellular Ca2++ in B. subtilis cells. Logarithmic phase cells expressing the apoaequorin were reconstituted with coelenterazine as described in Materials and Methods. Cells were washed with buffer A and loaded onto 96-well microtiter plate. Luminescence was read every second for 90 seconds. CaCl2 was injected at the time indicated by the arrow. For EGTA and LaCl3 treatments, cells were pre-incubated with EGTA (0.5 mM) for 15 min or the Ca2++ channel blocker LaCl3 (200 μM) for 30 min. Cells lacking the aequorin plasmid were used as controls. A) CaCl2 was injected to give the following concentrations: 0.5 mM, 1 mM, 5 mM and 15 mM. B) Effect of preincubation with EGTA or LaCl3. Cells preincubated with 0.5 mM EGTA (■), 200μM LaCl3 (▲), cells expressing aequorin, no treatments (♦) and control cells lacking the apoaequorin plasmid (■). |
