M5670

Dorsal hippocampi were collected around the tips of the hippocampal cannulas. Tissue was lysed in modified RIPA buffer, incubated 15 min on ice, and centrifuged for 15 min (15,000g) at 4 °C. Samples were subjected to SDS-PAGE (10 μg per well) and transferred to PVDF membrane (Millipore). Membranes were blocked with I-block (Tropix), incubated with primary antibody overnight at 4 °C, and with secondary antibody for 1 h at 20–22 °C. Bands were detected using alkaline phosphatase chemiluminescence. Primary antibodies used were against β-tubulin (1:4,000, T4026, Sigma), KCC2 (1:2,000, MABN88, Millipore), GABRA4 (1:1,500, sc-20917, Santa Cruz), GABRB2 (1:1,500, ab8340, Abcam), pPKCα/β II (1:800, T638/641), pPKCβ II (1:800, S660), pPKCθ (1:800, T538), pPKCδ/θ (1:800, S638/676) (PKC Sampler kit #9921, Cell Signaling), synapsin-2 (1:1,000, NB 120-13258, Novus Biologicals), NR1 (1:6,000, sc-9058, Santa Cruz), NR2A (1:1,000, 07-632, Millipore), NR2B (1:2,000, ab65783, Abcam), PKA (1:20,000, ab76238, Abcam), CaMKII (1:6,000, C6974, Sigma), Erk-1/2 (1:4,000, M5670, Sigma). All antibodies gave bands at the predicted molecular sizes and were validated by preadsorption experiments with immunogenic peptides used for their production. We also validated the Erk and NR2A antibodies with tissue of knockout mice (data not shown).

S2 cells were collected into an ice-cold microcentrifuge tube containing 1 ml of anticoagulant buffer and immediately washed twice with anticoagulant buffer. Washed cells were solubilized by adding same volume of 2 × lysis buffer (100 mM HEPES-NaOH, 300 mM NaCl, 3 mM MgCl₂, 1 mM EDTA, 20% glycerol, 2% TX-100 (w/v), 1% sodium deoxycholate, 0.2% SDS, pH 7.5) containing Protease inhibitor cocktail (Nacalai tesque, Japan), phosphatase inhibitor cocktail set II (Calbiochem, USA), and 0.2% phenylthiourea. After centrifugation at 17,000 g for 15 min at 4 °C, the supernatant was mixed with the same volume of sample buffer (125 mM Tris–HCl, 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue) and separated by SDS–PAGE (8 or 10%), transferred onto an Immobilon-P PVDF membrane (Millipore, USA). Proteins on the membrane were probed with anti-activated MAP Kinase (diphosphorylated ERK-1&2) mouse monoclonal antibodies (at 1:2,000, M9692, Sigma-Aldrich, USA)^{38 (link)}. For reprobing, membranes were washed for 30 min at 50 °C in 62.5 mM Tris–HCl (pH 6.7) containing 100 mM 2-mercaptoethanol and 2% SDS, and they were probed with anti-MAP kinase (ERK-1&2) mouse monoclonal antibodies (at 1:2,000, M5670, Sigma-Aldrich, USA). All positive bands were quantified using ImageJ (NIH).