Figure 3.

Biochemical and Biophysical Analysis of ERK2-Sub-D Interactions

(A and B) Steady-state kinetics analysis of ERK2 toward Sub-D following pre-treatment of ERK2 with 2.5 μM H₂O₂ (red) or dH₂O (untreated; blue). In (A), the concentration of Sub-D was varied while holding ATP constant at 20 μM while in (B), Sub-D was held constant at 1 μM while varying ATP levels. Error bars represent standard error about the mean (n > 3 independent experiments repeated in duplicate). Those points that exhibited a statistically significant difference in initial velocity (v₀) between treated and untreated samples are indicated by an asterisk (∗; p < 0.05; ∗∗p < 0.01).

(C) Representative equilibrium binding experiment (from among 3 independent experiments done in duplicate) measuring the affinity of oxidized ERK2 for Lig-D using surface plasmon resonance (SPR) at Lig-D concentrations ranging from 1.6 to 100 μM. The binding curve is shown in the inset.

(D) Average K_D,app for untreated ERK2 (blue) and ERK2 treated with 2.5 μM H₂O₂ before immobilization (n = 3 independent experiments repeated in duplicate). Error bars represent standard error about the mean. Statistically significant differences are indicated by an asterisk (∗, p < 0.05).