(a) Schematic diagram showing the experimental set up. CaMKII was activated, followed by wash to remove the components of the activation buffer and then saturating amounts of λ-phosphatase were added for 3, 15, or 30 min, in the presence and absence of Ca2+/CaM. (b) Plot showing the fraction of CaMKII-α holoenzymes with detectable phosphorylation at the activating site (Thr 286, right panel) after 0 min (activated control) and 3, 15, or 30 min of treatment with saturating amounts of λ-phosphatase in the absence (green trace) and presence (pink trace) of Ca2+/CaM. The fractions at 3, 15, or 30 min are normalized with respect to activated CaMKII that has not been exposed to any λ-phosphatase (0 min, whose value is set to 1.0). (c) Intensity distribution for pThr 286 (561 nm) for CaMKII-α holoenzymes with detectable phosphorylation, upon 0, 3, 15, or 30 min of λ-phosphatase treatment in the presence of 5 μM Ca2+/CaM (see Materials and methods for details of normalization).