FIGURE 5.

Role of ERK1/2 phosphorylation in preconditioning. A, cortical neuron cultures were subjected to 48-h low 4-AP/bic preconditioning (light bars) or to control wash (dark bars), in the absence (untreated) or presence of a pERK1/2 inhibitor, U0126 (20 μm; n = 12), followed by a 24-h recovery and then exposure to 65–80 min OGD, and measurement of the % PI-uptake 24 h later. B, to monitor neuronal Ca²⁺ spiking, cultures were loaded with 4.5 μm Fluo-4-AM, treated with low 4-AP/bic in the absence or presence of U0126 for 0.5–2.5 h, and fluorescence images were acquired for 120 s. C, representative pCREB and CREB immunoblots and densitometric analyses of pCREB:CREB ratios from n = 4 experiments following a 48-h treatment with control wash or low 4-AP/bic in the presence or absence of U0126 (20 μm). D, representative pERK1/2 and actin immunoblots and densitometric analyses of pERK1/2: actin ratios from n = 4 experiments for protein samples collected following a 6-h treatment with control wash or low 4-AP/bic in the presence or absence of TTX (1 μm), memantine (12.5 μm), MK-801 (2.5 μm), or NBQX alone (10 μm). E, representative pERK1/2 and actin immunoblots from n = 4 experiments and densitometric analysis of pERK1/2:actin ratios following a 6-h treatment with control wash or low 4-AP/bic ± MK-801 (2.5 μm), nifedipine (5 μm), MK-801 plus nifedipine, or nifedipine plus memantine. Brackets denote a significant difference (p < 0.05) between indicated conditions using an ANOVA.