Validation of interaction between synaptotagmin I and Kv1.4 channel. (A) Silver-stained SDS-PAGE of the protein complexes affinity purified from rat hippocampal plasma membrane-enriched fraction either with a Kv1.4-specific antibody (anti-Kv1.4) or a preimmunisation IgG pool (Pre-IgG). Arrowheads denote the bands identified by nano-LC tandem mass spectrometry as Kv1.4 and synaptotagmin I, respectively. (B) Western blot showing reverse purification of Kv1.4 from the same fraction with anti-synaptotagmin I. (C) Immunohistochemical analysis of the colocalization of synaptotagmin I and Kv1.4 in rat hippocampus. (a)-(c) show the localizations of pyramidal cell nuclei, Kv1.4 and synaptotagmin I in the CA2- CA3 regions, respectively. (d)-(f) show the localizations of pyramidal cell nuclei, Kv1.4 and synaptotagmin I in the CA1 region and denote gyrus, respectively. (g)-(i) are the enlarged images of CA3 region showing the localizations of Kv1.4 (g), synaptotagmin I (h) and their colocalization (i) in this region.