Figure 1. Oxotremorine-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/Gα15/M2 cells. The cells were loaded with Calcium-4 prior to being stimulated with an M2 receptor agonist, Oxotremorine. The intracellular calcium change was measured by FLIPR^TETRA. The relative fluorescent units (RFU) were normalized and plotted against the log of the cumulative doses (10-fold dilution) of Oxotremorine (Mean ± SD, n = 2). The EC₅₀ of Oxotremorine on this cell was 63.7 nM.
Notes:
1. EC₅₀ value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom)/(1+10^((LogEC₅₀-X)*HillSlope))
X is the logarithm of concentration. Y is the response
Y is RFU and starts at Bottom and goes to Top with a sigmoid shape.

Figure 2. 10 μg of membranes prepared from CHO-K1 cells stably expressing M2 receptors were incubated with indicated concentrations of [³H]N-Methylscopolamine ([³H]NMS) in the absence (total binding) or presence of 1000- fold access unlabeled Atropine (nonspecific binding, NSB). Binding was terminated by rapid filtration. Specific binding was defined by subtracting NSB from total binding. Data were fit to one-site binding equation using a non-linear regression method.

Figure 3. 10 μg of membranes prepared from CHO-K1 cells stably expressing M2 receptors were incubated with indicated concentrations of Atropine in the presence of 0.2 nM [³H]N-Methylscopolamine ([³H]NMS). Binding was terminated by rapid filtration. Data were fit to one-site competition equation using a non-linear regression method.