[Google Scholar] 28. mossy fiber pathways, and in the globus pallidus and substantia nigra. K252a Kv2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these -subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kv1 and Kv2 associate and colocalize with Kv1 -subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 – and -subunits to electrophysiologically diverse neuronal K+ currents. All reagents were molecular biology grade from Sigma (St. Louis, MO) or Boehringer-Mannheim (Indianapolis, IN), except where noted otherwise. The production of the anti– and anti–subunit-specific monoclonal and affinity-purified polyclonal antibodies is described in detail elsewhere (Trimmer, 1991;Rhodes et al., 1995, 1996; Bekele-Arcuri et al., 1996; Shi et al., 1996). In brief, these antibodies were raised using synthetic peptides or fusion proteins as the immunogen. Each antibody was examined for specificity on immunoblots of rat brain membranes, on immunoblots of membranes prepared from COS-1 cells transiently transfected with a broad panel of K252a K+ channel cDNAs, and by K252a immunofluorescence staining of transiently transfected cells (Bekele-Arcuri et al., 1996). Each antibody recognized only the appropriate protein on these immunoblots and stained only cells transfected with the appropriate cDNA. Moreover, immunoreactivity was completely eliminated by previous incubation with the corresponding peptide or fusion protein immunogen. A crude adult rat brain synaptosomal membrane fraction was prepared as described previously (Trimmer, 1991;Rhodes et al., 1995). Immunoprecipitation reactions were performed at 4C using detergent lysates of these membranes as described previously (Rhodes et al., 1995, 1996). In brief, membranes (1 mg of membrane protein/tube) were solubilized to 1 1 ml final volume per tube in lysis buffer [1% Triton X-100 and (in mm) 150 NaCl, 1 EDTA, 10 sodium azide, and 10 Tris-HCl, pH 8.0] containing a protease inhibitor mixture (Trimmer, 1991). Affinity-purified antibodies were added, and the volume was adjusted to 1 1 ml with lysis buffer. Samples were incubated for 2 hr on a rotator, followed by addition of 50 l of a 50% slurry of protein ACSepharose and further incubation for 45 min. After incubation, protein ACSepharose was pelleted by centrifugation at 10,000 for 20 sec, and the resulting pellets were washed by resuspension and centrifugation six times with lysis buffer. The final pellets were resuspended in 200 l of reducing SDS sample buffer. Products of immunoprecipitation reactions (20 l, representing the yield from 100 g of starting crude rat brain membrane protein) were size fractionated on 9% (for analysis of -subunit polypeptides) or 12% (for analysis of -subunit polypeptides) SDS-polyacrylamide gels (Maizel, 1971). Sixty micrograms of crude rat brain membrane protein were also resuspended in reducing SDS sample buffer and loaded directly onto each SDS gel. Disulfide bonds were reduced by the addition of 20 mm 2-mercaptoethanol to the sample buffer. Lauryl sulfate (Sigma) was the SDS source used for all SDS-PAGE (Shi et al., 1994). After electrophoretic transfer to nitrocellulose paper, the resulting blots were blocked in TBS containing 4% low-fat milk (Blotto) (Johnson et al., 1984), incubated in affinity-purified antibody diluted 1:50C1:2000 in Blotto for 1 hr or undiluted monoclonal antibody tissue culture supernatants, and washed three times in Blotto for 30 min total. Blots were then incubated in HRP-conjugated secondary antibody (Organon Teknika, West Chester, PA; 1:2000 dilution in Blotto) for 1 hr and then washed in TBS three times for 30 min total. The blots Ptprc were then incubated in substrate for enhanced chemiluminescence (ECL) for 1 min and autoradiographed on preflashed (to OD545 = 0.15) Kodak (Rochester, NY) XAR-5 film. The procedures for single-label light microscopic immunohistochemistry are described in detail elsewhere (Rhodes et al., 1995, 1996; Bekele-Arcuri et al., 1996). K252a Briefly, 35-m-thick sections of adult rat brain were incubated overnight at 4C in an antibody vehicle containing affinity-purified rabbit polyclonal or mouse monoclonal antibodies. Detection of antibodyCantigen complexes was accomplished using the avidinCbiotin ABC procedure (Vector Laboratories, Burlingame, CA) and visualized using a nickel-enhanced diaminobenzidine procedure (Rhodes et al., 1995, 1996). For multiple-label.
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