mM Na2EDTA, 5 mM DTT, 0.01% Brij35 at 30uC for 10 min. The reaction was terminated by adding TCA followed by centrifugation. The amount of free phosphates released in the reaction was purchase 863774-58-7 measured with BIOMOL Green reagent at 620 nm and background-subtracted. For total phosphatase activity, tautomycin and OA were removed from the reaction. PP1 and PP2A activity was calculated using the ratio of phosphatase activity with inhibitors and total phosphatase activity. Electrophysiology Three to four month old Cdk5f/f/T29 mice or their Cdk5f/f control littermates were sacrificed by cervical dislocation, and hippocampi were rapidly dissected in ice-cold oxygenated artificial CSF. Transverse hippocampal slices were placed in a chamber and continuously perfused with oxygenated ACSF consisting of: 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 11 D-glucose, pH 7.4. A bipolar stimulating electrode placed in the 
stratum radiatum was used to elicit action potentials in CA3 Schaffer collateral axons. An ACSF-filled glass microelectrode with a resistance between 0.5 and 3 MV was placed in the stratum radiatum region of area CA1 and was used to record the field excitatory post-synaptic potentials. Data were acquired using HEKA EPC10 and analyzed by Patchmaster. Peak fEPSP amplitudes from stimulators were required to be at least 2 mV, and stimulus intensity was set to produce 40% of the maximal response. Baseline responses were recorded for 20 min. fEPSPs were evoked at area CA1 synapses by stimulating Schaffer collaterals at a low frequency to establish a stable baseline. Immediately following LTP induction with 2 trains of high-frequency stimulation, with an interval of 20 s, slices from Cdk5f/f/T29 and control Cdk5f/f mice showed an increase in fEPSP slope and amplitude, suggesting that short-term potentiation occurs in all groups. PSD preparation Forebrains from Cdk5f/f and Cdk5f/f/CW2 mice were homogenized and postsynaptic densities isolated in ice-cold buffers with protease and phosphatase inhibitor cocktails as previously described with minor modifications. Briefly, mouse forebrains were isolated and homogenized in ice-cold Buffer A, 1 mM MgCl2, 0.5 mM CaCl2) with a Teflon homogenizer. Homogenized brain extracts were spun at 14006g for 10 min. The supernatant was saved and the subsequent pellet was homogenized again. After centrifugation at 7006g, the supernatant was saved and pooled with S1. Pooled S1 and S1′ was centrifuged at 13,8006g for 10 min to collect the pellet. ” P2 was resuspended in Buffer B. The P2 suspension was loaded onto a discontinuous sucrose gradient, followed by centrifugation for 2 h at 82,5006g in a SW-41 rotor. The synaptosome fraction between 1 M and 1.15 M sucrose was collected with a syringe needle and adjusted to 4 ml with Buffer B. Equal volumes of Buffer C was added and mixed for 15 min and then spun at 32,8006g in a Ti70.1 rotor for 20 min. The PSD-enriched pellet was resuspended in 40 mM Tris and protein concentration was measured using a BCA assay. To prepare samples for mass spectrometry, solubilized PSD proteins were reduced with 10 mM DTT for 30 min at 60uC. After cooling to room temperature, the sample was then alkylated 13 September 2011 | Volume 6 | Issue 9 | e25735 Phosphatase assay Whole hippocampi were dissected and homogenized in 3.75 mM Tris-HCl, pH 7.4, 15 mM “ 25331948 KCl, 3.75 mM NaCl, 250 mM EDTA, 50 mM EGTA, 30% sucrose, 30% glycerol, protease inhibitor cocktail, 100 mM PMSF using a Dounce homoge
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