TIRFM responses to prolonged stimulation with 10 mM Ca²⁺.

<p><b>A.</b> HEK293 cells expressing BS-wt were stimulated with 10 mM Ca²⁺ in the absence (closed circles) or presence of tunicamycin (open circles) for 30 min, followed by return to 0.5 mM Ca²⁺. Plasma membrane SEP fluorescence was monitored throughout the time course by TIRFM as described in Methods. Data are average ± S.D. of 4 cells. <b>B.</b> Experiment as in <b>A</b> for cells expressing BS-5A mutant. Data are average ± S.D. of 4 cells. <b>C.</b> Cells expressing BS-wt were stimulated with 0.5 or 10 mM Ca²⁺ for 60 min, 37°C, followed by fixation, permeabilization, and staining with anti-GFP antibody (secondary antibody tagged with Alexa-568). Confocal images of SEP and anti-GFP fluorescence are shown along with the merged image. <b>D.</b> Experiment as in <b>C</b> for cells expressing BS-5A mutant.