Fig. (4) Using the optimal concentration of unactivated ERK2 of 140 nM (~10 µg/ml) and unactivated MEK1 of 20 nM (~1 µg/ml), determination of amount of active B-RAF V599E to use for subsequent kinase inhibitor studies (♦, triple cascade). Control reactions lacking a portion of the RAF-MEK-ERK pathway were: titration of B-RAF V599E without MEK1 and without ERK2 (□); titration of B-RAF V599E with 140 nM unactivated ERK2 without MEK1 (▽); titration of B-RAF V599E with 20 nM unactivated MEK1 without ERK2 (×). (D) Linear plots of the percent phosphorylation achieved for each of the triple cascades with increasing concentrations of active RAF isoform, using the optimized concentrations for unactive MEK1 (20 nM) and unactive ERK2 (140 nM), with R² ≥ 0.99. All data points represent the mean ± standard deviation from triplicate samples.