Figure 4.

Ras-GRF1 and Ras-GRF2 make different contributions to NMDA-induced Erk activation via different classes of NMDARs in the hippocampus of postpubescent mice. A, Hippocampal brain slices from mature (> P25) double Ras-GRF knock-out mice, as well as genetically matched control mice, were treated with NMDA (100 μm) for various times. Then the slices were lysed in detergent, and immunoblots of lysates were performed by using activation-specific phospho-Erk1/2 antibodies or antibodies to total Erk1/2. Quantification of the fold increase in Erk1/2 activation after NMDA stimulation also is shown (n > 3; ± SD, **p < 0.01). B, Similar analysis was performed on hippocampal brain slices from P14–P18 control and double Ras-GRF knock-out mice except that only the 5 min time point is shown. The data are representative of at least two independent experiments. C, Control, grf1(−/−), and grf2(−/−) knock-out mice (> P25) were analyzed along with double grf1/grf2(−/−) mice as described in B. No significant difference in NMDA-induced Erk activation was observed among control strains used for grf1(−/−), grf2(−/−), or double knock-out mice, so a single value of pooled controls was used to represent control samples from wild-type (WT) mice. Band intensities of phospho-Erk1/2 were normalized to the band intensity of total Erk (n ≥ 4; ± SD; **p < 0.01). D, Hippocampal brain slices from mature (> P25) ras-grf1(−/−) mice were treated with NMDA for 5 min as described in A. In some samples the brain slices were pretreated with ifenprodil (IF) (5 μm) for 30 min or NVP-AAM077 (NVP; 0.6 μm) for 50 min before NMDA stimulation. Erk activation was detected, and the fold increase was quantified as above (n ≥ 4; ± SD; **p < 0.01). E, Similar experiments were performed as in A except that the hippocampal brain slices were prepared from adult ras-grf2(−/−) mice (n ≥ 4; ± SD; **p < 0.01).