134813.fig.007a
(a)
134813.fig.007b
(b)
Figure 7: The binding of hCaMKP-N(1–559) to PSD and the dephosphorylation of the autophosphorylated CaMKII. (a) hCaMKP-N(1–559) (1 μg) was incubated on ice for 1 h with (lane 2) or without (lane 3) the PSD fraction (1 μg) as described in Section 2. After incubation, the mixture was centrifuged, and the pellet fraction was washed twice with 50 mM Tris-HCl (pH 7.5) containing 0.85% NaCl, followed by western blotting analysis using anti-CaMKP-N antibody (upper panel). Recovery of the PSD fraction was confirmed by probing the same blot using anti-CaMKIIα antibody (lower panel). To check the endogenous CaMKP-N levels, the PSD fraction was also incubated in the absence of hCaMKP-N(1–559) as a control (lane 1). (b) The PSD fraction (29 μg/mL), in which CaMKII had been autophosphorylated as described, was incubated at 30˚C with (lane 2) or without (lane 1) hCaMKP-N(1–559). After incubation for 30 min, the phosphatase reaction was terminated by adding excess EDTA (20 mM), and aliquots were analyzed by western blotting to examine the extent of phosphorylation at Thr286 on CaMKII (upper panel, anti-PCaMKII) and the total amount of CaMKIIα on the blot (lower panel, anti-CaMKIIα). The data presented are representative of at least three independent experiments with similar results.