Figure 4.

Greater association of GluN2B with RasGRF1 occurs under conditions that show GluN2B involvement in LTD. A,B) Sagittal hippocampal slices were prepared with the same methodology as for the electrophysiological experiments and treated with control aCSF (C) or 20 μM scopolamine in aCSF (S) for 2 hrs, followed by homogenization in RIPA buffer. 500 μg of each lysate was used for immunoprecipitation with a polyclonal GluN2B antibody bound to protein A sepharose. After washing in RIPA, the coIPs were subjected to SDS PAGE and Western blot, alongside 30 μg per lane of the crude lysates. The blots were sequentially probed with RasGRF1 and GluN2B antibodies using HRP- and AP-conjugated secondary antibodies respectively. In each coimmunoprecipitate the level of RasGRF1 was normalized to GluN2B and then a scopolamine/control ratio was calculated and averaged across experiments. C, D) Coronal (C) or sagittal (S) slices were prepared and recovered in the absence of scopolamine. The lysates were processed and taken forward into immunoprecipitation as described above.