Table 3.

Table showing the inputs for fitting the set of Eqs. (9), (17), and (18) in GraphPad Prism software. Columns include parameter definitions, constraints on the fit parameters, initial value predictions for each parameter, and the results of the fit. FA = fluorescence anisotropy. Numerical values shown in the table are unitless unless otherwise noted as micromolar concentrations.

Parameter	Description	Constraint	Initial value	Fit result
Kd	The dissociation constant for FITC-X-Lig-D binding	=1.5 (μM)	Fixed (KD from Eq. (11))	1.5 (μM)
St	Total ligand concentration	=0.010 (μM)	Fixed	0.010 (μM)
rb	FA signal for maximally bound complex	Must be greater than 0	“YMAX” (maximum FA value)	226 ± 6
rf	FA signal for 100% free ligand	Must be greater than 0	“YMIN” (minimum FA value)	55 ± 1
R	Fluorescence intensity correction factor	=1.8	Fixed (R from Eq. (5))	1.8
Et	Total ERK2 concentration	=1.0 (μM)	Fixed	1.0 (μM)
Ki	The dissociation constant for inhibitor binding	Must be greater than 0	1.3 (μM) ($I{C}_{50}$ from Eq. (12))	0.18 ± 0.04 (μM)