Figure 2. Slight reactivation of phosphatase-treated ERK8 after incubation with MgATP.

(A) Wild-type GST–ERK8 (grey bars) or His₆–ERK8 (black bars) were incubated in the absence or presence of PP2A plus PTP1B as in Figure 1(B), and the phosphatases were then inhibited by the addition of the PP2A inhibitor microcystin–LR (0.1 μM) and the PTP1B inhibitor sodium orthovanadate (0.1 mM). The untreated or inactivated ERK8 were then incubated with MgATP, and aliquots of the reactions were removed at the times indicated and assayed for activity using MBP as a substrate. (B) Further aliquots from (A) were spotted on to nitrocellulose membranes and immunoblotted as in Figure 1. NP, no phosphatase; ERK8 not pre-treated with PP2A plus PTP1B and incubated for 6 h without MgATP.