Fig. 1. Reciprocal co-immunoprecipitation from rat brain and hippocampus demonstrated selective co-assembly of BK and N-type Ca²⁺ channels. Western immunoblots of proteins isolated from soluble whole brain (A,C) or hippocampal (B) extracts by immunoprecipitation using antibodies for the -subunits of the BK channel (anti-BK_1118-1135; BK-IP) or the Ca_V2.2 channel (anti-Ca_V2.2; Ca_V2.2-IP). (Ai) Probing BK-IP and rabbit IgG IP (IgG-IP) samples with anti-Ca_V2.2 revealed a band of 210 kDa in the BK-IP sample lane but not in the IgG control co-IP lane, indicative of the lower molecular mass form of the Ca_V2.2-subunit (n=4). (Aii) Enrichment of the immunoreactive band for the BK channel -subunit (120 kDa) in the BK-IP sample demonstrated the specificity of the immunoprecipitation. (B) The Ca_V2.2-subunit was co-immunoprecipitated with the BK channel -subunit from solubilised rat hippocampal tissue. This was seen as a band of 210 kDa in the BK-IP sample, which was absent in the control IgG-IP lane. (Ci) Probing Ca_V2.2-IP and rabbit IgG co-IP (IgG-IP) samples with anti-BK produced an immunoreactive band of the predicted molecular mass of BK channel -subunit (not observed in the IgG control co-IP lane), showing that the BK channel -subunit reciprocally co-immunoprecipitated with the Ca_V2.2-subunit (n=3). (Cii) Enrichment of the immunoreactive band for the Ca_V2.2-subunit (210 kDa) in the Ca_V2.2-IP sample demonstrated the specificity of the immunoprecipitation. In each of the above, a solubilised whole brain extract (input) was run alongside the co-immunoprecipitation samples. Input was 5% of total protein extract used in the assay. The positions of channel proteins and the heavy chain IgG (HC IgG) of the immunoprecipitating antibodies are indicated by arrows, and molecular mass standards are shown in each immunoblot.