The anti-Sur1 and anti-Trpm4 antibodies used in this study are listed in Table 2. Anti-Myc and anti-HSC70 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG antibody and anti-calmodulin antibody were purchased from Cell Signaling Technology (Beverly, MA). Two different sources of both anti-Sur1 and anti-Trpm4 antibodies were developed for this study (Table 2). To construct a bacterial expression vector encoding the intracellular nucleotide-binding domain 1 of Sur1 fused with hexahistidine, the corresponding region of the rat Sur1 cDNA sequence (amino acids 598–965 of NP_037171) was cloned into pQE30 (Qiagen, Gaithersburg, MD). The recombinant protein was purified by using a Ni+-nitrilotriacetic acid-agarose column and used to raise antibodies in rabbit and in goat, which was performed by a commercial service (Covance, Princeton, NJ). Using the same method, anti-Trpm4 antibodies targeting the N-terminal intracellular domain of mouse Trpm4, corresponding to amino acids 1–612 (NP_780339), were developed in rabbit and in chicken.
The specificity of the antibodies described above was validated using lysates from the appropriate Sur1 or Trpm4 expression systems and spinal cord tissues from wild-type, Trpm4−/−, and Abcc8−/− mice. Anti-Sur1-b antibody and anti-Trpm4-b antibody were used to immunoisolate Sur1 and Trpm4, respectively, from the various lysates. Subsequently, in one experiment, immunoblots of the immunoisolated proteins were performed using anti-Sur1-a antibody and anti-Trpm4-a antibody, respectively (see Fig. 9). In a parallel experiment, electrophoresis gels of the immunoisolated proteins were developed with Coomassie Blue stain, the protein bands at the appropriate molecular masses were isolated, and peptide fragments were analyzed by mass spectrometry (Taplin Mass Spectrometry Facility, Harvard Medical School, Boston, MA), which confirmed the identity of Sur1 and Trpm4.