Fig. 7.

In vivo and in vitrointeractions between GluR-D and 4.1N. A, In vivo interaction of 4.1 and GluR-D-containing AMPA receptors. Rat cerebellar detergent extract was immunoprecipitated with the antisera or preimmune sera indicated above. Immunoblots were probed with the antisera indicated to the left, followed by anti-rabbit IgG conjugated to HRP. B, HEK293 cells were either cotransfected with F-GluR-D and Myc-4.1N (Co-T) expression vectors or singly transfected with the constructs; the latter were mixed during solubilization and immunoprecipitation (Mixed). The extracts were then immunoprecipitated with antibodies to the Flag and Myc tags, as indicated above. Immunoblots were probed with the antibodies indicated to the side. C, Interaction of 4.1N protein with the CTDs of GluR-A, GluR-B, GluR-C, and GluR-D. HEK293 cell extract expressing Myc-4.1N was probed by GST pull-down assay using the fusion proteins indicated above. The blot was then probed by anti-Myc IgG, followed by anti-mouse IgG-HRP. The bandsseen with the GluR-B and GluR-C GST fusion proteins correspond to background levels seen with GST alone. D, The interaction of 4.1N with GluR-D requires the M4 proximal region. TheInput panel shows the expression of Myc-tagged 4.1N in the lysate of transfected HEK293 cells. The right-hand panel shows the precipitation of the expressed Myc-4.1N by the GST fusion proteins indicated above. Both panels were probed by anti-Myc IgG, followed by anti-mouse IgG-HRP. The bottom panel shows Ponceau S staining of the expressed GST fusion proteins used in the pull-down assay.