Western blot analysis was performed as described previously (Spencer et al. 2003). Neurons cultured in six-well plates or 92 × 17 mm petri plates were placed in a water bath at 37°C and left for 5 min to equilibrate. After this period, the culture medium was removed and replaced with HEPES-buffered incubation medium (HBM; 20 mmol/L HEPES (pH 7.4), 140 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L NaHCO3, 5 mmol/L Na2HPO4, 1.2 mmol/L CaCl2 and 5.5 mmol/L glucose). Neurons were pre-incubated for 5 min with kinase inhibitors (MAP kinase kinase (MEK)1/2 inhibitor U0126 or phosphatidylinositol (PI)3-kinase inhibitor LY294002), or vehicle, prior to treatment with flavonoids. Following pre-incubation, neurons were stimulated for 15 min with (-)epicatechin or (-)epicatechin metabolites (30 nmol/L–30 μmol/L) in the further presence or absence of kinase inhibitors where appropriate. After 15 min, the HBM was removed and for the measurements of Akt, pAkt, CREB, pCREB ERK and pERK, the plates were quickly washed with ice-cold phosphate buffered saline (pH 7.4) (Ca2+-free with 200 μmol/L EGTA) and placed immediately on ice. For measurement of GluR2, GluR1 and ERK expression, the HBM was removed and replaced with neuronal conditioned medium and the cells returned to the CO2 incubator for a further 18 h. Crude lysates were prepared by scraping the cell monolayer in ice-cold 1% Triton X-100 lysis buffer (50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl and 1% Triton X-100) containing a cocktail of inhibitors at a final concentration of 2 mmol/L EDTA, 2 mmol/L EGTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL antipain, 1 μg/mL pepstatin A, 1 μg/mL chymostatin, 5 mmol/L sodium pyrophosphate, 1 mmol/L Na3VO4 and 50 mmol/L NaF, and left on ice for 45 min before centrifugation at 2000 × g at 4°C for 5 min. The protein concentration in the supernatants was determined using Bio-Rad protein assay reagent (Hemel-Hempstead, UK) and samples were then denatured by boiling for 5 min in gel loading buffer [62.5 mmol/L Tris (pH 6.8), 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol, 10% glycerol and 0.0025% bromophenol blue]. Samples were run (20 μg/lane) on 9% sodium dodecyl sulphate–polyacrylamide gels and transferred to nitrocellulose membrane by electroblotting. Immunoblots were then blocked [20 mmol/L Tris (pH 7.5), 150 mmol/L NaCl] (TBS) containing 5% skimmed milk powder for 30 min washed for 10 min in TBS supplemented with 0.05% (v/v) Tween 20 (TTBS) and then incubated with primary antibodies overnight at 25°C in TTBS containing 1% skimmed milk powder at the following dilutions: anti-ACTIVE MAPK (1 : 5000), ERK1/2 (1 : 1000), phospho-CREB (1 : 2000), CREB (1 : 1000), phospho-Akt (1 : 2000), Akt (1 : 1000), GluR2 (1 : 1000) or GluR1 (1 : 1000). The immunoblots blots were washed twice in TTBS and then incubated in TTBS containing 1% skimmed milk powder with goat anti-rabbit IgG conjugated to horseradish peroxidase (1 : 1000 dilution of stock) for 1 h. Finally, immunoblots were washed in TTBS for 5 min, rinsed in TBS for 10 min and then treated with ECL reagent. Immunoblots were then exposed to Hyperfilm ECL for 1–2 min and developed. Relative bands intensities were obtained by densitometric analysis of films using Bioimage Intelligent Quantifier software (Ann Arbor, MI, USA). Molecular weights were calculated from comparison with pre-stained molecular weight markers (MW 29 000–205 000).