Total camkii

Luminex xMAP multiplexed immunoassays (Millipore, Billerica, MA) were used to evaluate the levels of phosphorylated proteins in the hippocampal formation and in primary neuron cultures. The hippocampal tissue or primary neurons were lysed with MilliplexMAP Cell Signaling Universal Lysis Buffer containing phosphatase inhibitors including 1 mM sodium orthovanadate and freshly prepared 1× protease inhibitor cocktail (EMD Millipore). The phosphoprotein analyses used were: CREB (Ser133), AKT (Ser473), Erk1/2 (Thr185/Tyr187), JNK (Thr183/Tyr185), p70 S6 Kinase (Thr412), p38 (Thr189/Tyr182), STAT3 (Ser727), STAT5 (Tyr694/699), and NFκB (Ser536). 20 µg protein lysate was used for the assays, according to the procedure provided by the manufacturer. For protein gel-blot analysis, the same samples were used for SDS-PAGE protein separation, and antibodies specific for phosphomTOR and total mTOR are from Millipore, phospho-p70S6K is from Cell Signaling, total CaMKII, phospho-CaMKII and β-actin were obtained from Santa Cruz Biotechnology.

The changes in the expression and activation of tau kinases were examined using the following antibodies: GSK-3β (1:1000, BD Transduction Lab, Franklin Lakes, NJ) as well as the following antibodies purchased from Cell Signaling Technology, Inc. (Danvers, MA): phospho-GSK-3β (Ser9, 1:1000), p35/p25 (1:1000), phospho-Akt (Ser⁴⁷³, 1:1000), Akt (1:1000), SAPK/JNK (1:1000), phospho-SAPK-JNK (Thr¹⁸³/Tyr¹⁸⁵, 1:1000), p44/42 MAPK (Erk 1/2, 1:1000), phospho-p44/42 MAPK (Erk 1/2, Thr²⁰²/Tyr²⁰⁴, 1:1000), p38 MAPK (P38, 1:1000). Cdk5 (C-8), phospho-Ca²⁺/calmodulin-dependent protein kinases II (CaMKII, Thr²⁸⁶, 1:1000) and total CaMKII (1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-p38 (clone 6E5.2, p-P38, Thr¹⁸⁰/Tyr¹⁸², 1/1000) was purchased from Millipore, and GSK-3 α/β PY279/PY216 (Tyr²⁷⁹/Tyr²¹⁶, 1:1000) from Life Technologies (Grand Island, NY).
The changes in the expression of the tau phosphatases were determined using antibodies directed at PP2A-C (1:1000, Cell Signaling), PP5 (1:1000, Cell Signaling), PP2A-Bα subunit (2G9), 1:1000, Cell Signaling), demethylated PP2A-C subunit (4B7, 1:1000, Santa Cruz), PP2A-A subunit (1:1000, Cell Signaling), pan-calcineurin A (PP2B, 1:1000, Cell Signaling), and the PP1 catalytic subunit (E-9, 1:1000, Santa Cruz).