Figure 8.

Representative kinetic plots for immunoprecipitated sGC obtained from MCF-7 cells. Cells were lysed after treatment for 5 min with ionomycin, thapsigargin and 2 mM CaCl₂, or vehicle control. Reactions were carried out at 37 °C for 10 min (−NO) or 3 min (+NO). Where included, DEA/NO (50 µM) was added just prior to measurement. Shown are the averages of duplicate measurements ± the range in measured values. The solid curves represent the nonlinear fit to the Michaelis-Menten equation. B. Immunoprecipitated sGC treated with Jurkat cell lysate and 250 nM Ca²⁺ is inhibited; inhibition is reversed by broad-range protein kinase inhibitor staurosporine. sGC was immunoprecipitated from transiently transfected MCF-7 cells and incubated with Jurkat T cell lysate with or without 250 nM Ca²⁺ and/or 1 µM staurosporine. Calcium and staurosporine were washed away and cGMP activity was measured. Where indicated, DEA/NO and YC-1 were added to a final concentration of 10 µM. Error bars represent the standard deviation from the mean of independent experiments (n = 3)