FIGURE 4.

SNX26 interacts with PSD-95 through PX and RhoGAP domains. A, schematic representation of SNX26 full-length and various deletion mutants used in the experiments. Abbreviation used is as follows: RhoGAP, Rho GTPase-activating protein. B, schematic representation of full-length PSD-95 and deletion mutants used in the experiments. C–E, HEK293T cells were co-transfected with GFP, GFP-SNX26, or GFP-tagged deletion mutants and HA-PSD-95 (C) or co-transfected with GFP, PSD-95-GFP, or GFP-tagged deletion mutants and HA-SNX26 (D). Twenty four hours after transfection, the cells were lysed, immunoprecipitated with anti-GFP antibody, and immunoblotted with anti-HA antibody. IP, immunoprecipitation; IB, immunoblot. Interaction of SNX26 with PSD-95 was further investigated to pin down binding domains. E, HEK293T cells were co-transfected with each construct indicated, lysed, and immunoprecipitated with anti-GFP antibody followed by immunoblotting with anti-HA antibody. F, SNX26 endogenously interacts with PSD-95 in rat brains. Brain lysates were immunoprecipitated with an anti-PSD-95 antibody or normal rabbit serum (NRS), and immunoblotted with an anti-SNX26 antibody. G, neurons were co-transfected at DIV 13 with PSD-95-GFP and mCherry-SNX26 and fixed at DIV 14. PSD-95-GFP and mCherry-SNX26 are co-localized at spines. Scale bar, 15 μm. H, endogenous SNX26 partially co-localizes with PSD-95 at dendritic spines. Cultured hippocampal neurons at DIV 14 were fixed and immunostained with either a specific SNX26 or a specific PSD-95 antibody, followed by either Alexa 488-conjugated donkey anti-goat (SNX26) or Texas Red-conjugated anti-mouse antibody (PSD-95). Arrowheads indicate dendritic spines where two proteins were co-localized. Scale bar, 7 μm.