Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: Summary of Slo1 Antibodies

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques:

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A, immunoblot analysis of brain membrane fractions from adult rats, and wild-type and Slo1-deficient mice. Proteins were fractionated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes and probed with mouse monoclonal (L6/60, 10 µg/ml) or rabbit polyclonal anti-Slo1 (Alomone, 1:500; Chemicon, 1:200), or mouse monoclonal anti-Kv2.1 (K89/41, TC supe 1:2) antibodies as noted. Numbers to left denote mobility of prestained molecular weight standards in kD.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Western Blot, SDS Page, Molecular Weight

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A: Immunoblot analysis of the effects of alkaline phosphatase (AP) digestion on adult rat brain Slo1. Adult rat brain membranes treated without (−) or with 0.1 U/ml AP (+) for 3 h at 37°C were separated on 7.5% SDS-PAGE and transferred to nitrocellulose membranes, then probed with anti-Kv2.1 (K89/41 mouse mAb, TC supe 1:2), anti-Kv1.4 (K13/31 mouse mAb, TC supe 1:2), anti-GluR1 (rabbit polyclonal antibody, Upstate 1:1000), or anti-Slo1 (L6/60, 10 µg/ml) antibodies. Slo1 exhibited AP- shifts from Mr ≈ 135 to ≈ 128 kD (bands 1 and 2 indicated on the right). Brain Kv2.1 also shifted in Mr upon AP treatment, whereas Kv1.4 and GluR1 did not. B: Immunoblot analysis of developmental rat brain membrane samples. L6/60 (10 µg/ml) immunostaining at postnatal day 2 (P2) revealed at least three distinct bands as indicated in the left margin (1, 135 kD; 2, 131 kD; 3, 124 kD), which changed in their relative proportions during postnatal development. A=adult, C=adult sample incubated 3 h at 37°C without AP, AP=adult sample incubated 3 h at 37°C with AP. Numbers to left denote mobility of prestained molecular weight standards in kD.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Western Blot, SDS Page, Immunostaining, Incubation, Molecular Weight

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A–C: L6/60 staining in adult rat hippocampus A: overview of L6/60 (0.6 µg/ml) immunoperoxidase staining in hippocampus. B, C: higher magnification images of the staining shown in A. B: stratum lucidum of the CA3 region of the hippocampus. C: magnified view of mossy fiber axons. Arrows in A–C highlight anatomical landmarks that specify the regions magnified. D–E: double label immunofluorescence labeling of brain sections from wild-type (D, WT) and Slo1-deficient (E, KO) mice. Brain sections from these mice were stained with L6/60 (24 µg/ml) mAb in red, and anti-Kv2.1 (KC rabbit polyclonal, 1:100) antibody in green. Images were taken from the hilus of the dentate gyrus and CA3 stratum lucidum. Scale bars, A:, 500 µm; B, C: 100 µm; D: 50 µm.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Staining, Immunoperoxidase Staining, Immunofluorescence, Labeling

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: Magnified view of stratum lucidum of CA3 region of hippocampus. A–C: somata and apical dendrites of CA3 pyramidal neurons were negatively stained with L6/60 (A, 24 µg/ml) as well as with anti-Kv1.4 antibody (B, K13/31 mouse mAb, TC supe 1:2), which overlapped in mossy fibers (C). D–F: in contrast, somatodendritic Kv2.1 (D, KC rabbit polyclonal antibody, 1:100) staining interdigitated with L6/60 staining (E), indicating the presence of Slo1 on synaptic terminals on the Kv2.1-positive apical dendrites of CA3 pyramidal neurons (F, overlay). Scale bars, 10 µm.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Staining

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: These photomicrographs show the pattern of immunoreactivity for the indicated subunits in the unoperated, control hemisphere (A–E) and operated hemisphere (F–J) of an animal that sustained a circumscribed unilateral ibotenic acid lesion. This lesion destroyed cells in the distal CA1 subfield, prosubiculum and subiculum, and also destroyed a central portion of the dentate gyrus. The entire CA3 and proximal CA1 subfield was spared by this lesion. This lesion greatly reduced the density of Slo1 (A, F; L6/60 TC supe 1:10), and Kv1.4 (B, G; K13/31 TC supe 1:10) in stratum lucidum of CA3, but did not affect binding of secondary antibody alone (C, H), nor staining for Slo1 (D, I; L6/60 TC supe 1:10), and Kv1.4 (E, J; K13/31 TC supe 1:10) in the terminal fields of striatal effernts to globus pallidus.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Binding Assay, Staining

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A: low magnification view of L6/60 (0.6 µg/ml) immunoperoxidase staining near the primary fissure. Note moderate to high levels of staining in the molecular layer relative to the granule cell layer, and intense staining at/near the Purkinje cell layer. B: higher magnification view of the area boxed in A, showing intense L6/60 staining in the Purkinje cell layer. The arrowheads point to a Purkinje cell soma, while the arrow points to a basket cell pinceau terminal onto a Purkinje cell initial segment. C–D: Double label immunofluorescence staining of brain sections from wild-type (C: WT) and Slo1-deficient (D: KO) mice. Brain sections from these mice were stained with L6/60 mAb (24 µg/ml) in red, and anti-Kv2.1 (KC rabbit polyclonal, 1:100) antibody in green. Images were taken from the Purkinje cell layer and reveal that staining in Purkinje cell somata, basket cell terminals, and the molecular layer is eliminated in the Slo1 knockout. Scale bars, A:, 100 µm; B, C: 500 µm; D: 10 µm.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Immunoperoxidase Staining, Staining, Immunofluorescence, Knock-Out

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: Confocal images of double label immunofluorescence staining. A–C: Slo1 and Kv1.2 localization in basket cell terminals. L6/60 (A: 24 µg/ml) staining overlaps with that for Kv1.2 (B: K14/16 mouse mAb, 16 µg/ml) in basket cell terminals (C: overlay); D–F: L6/60 (D: 24 µg/ml) staining encircles axon initial segments of Purkinje cells, stained with anti-NF-155/186 antibody (E: L11A/41 mouse mAb, TC supe 1:2; F: overlay). G–H: dendrites of multiple Purkinje cells, filled with anti-calbindin (H: Sigma mouse monoclonal, 16 µg/ml) staining, were associated with membrane-associated L6/60 staining (G: 24 µg/ml; I: overlay). J–L: localization of Slo1 in Purkinje cell somata. Slo1 staining (J: L6/60; 24 µg/ml) on the somata of Purkinje cells partially overlapped with Kv2.1 (K: KC rabbit polyclonal antibody, 1:100) surface clusters (L: overlay). Scale bars, 10 µm.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Immunofluorescence, Staining

Journal:

Article Title: Immunolocalization of the Ca 2+ -Activated K + Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

doi: 10.1002/cne.20931

Figure Lengend Snippet: A–C: Localization of endogenous Slo1. Neurons at 24 DIV were fixed, permeabilized with 0.1% Triton X-100 and stained with anti-Slo1 antibody L6/60 (A: 24 µg/ml; green) to detect the total pool of endogenous rSlo1, and anti-MAP2 (B: Sigma mouse mAb, 1:1000; C: overlay). D–F: Localization of surface hSlo1. Neurons at 11 DIV were fixed, stained with anti-Myc antibody (E: mouse monoclonal 1-9E10, 1 µg/ml) for cell surface hSlo1 (green), and then permeabilized with 0.1% Triton X-100 for L6/60 (D: 5 µg/ml) staining (red) to detect the total pool of exogenous hSlo1 and endogenous rSlo1 (F: overlay). Arrow points to a presumably untransfected neuron in the culture. Panel D inset: hSlo in both axons and dendrites was detected on a longer exposure. G: Surface Myc-positive (mouse mAb 19E10, 1 µg/ml) processes overlap with axonal tau (Sigma mouse monoclonal antibody 1:2000) staining. H–J: Surface Myc-positive (H: mouse mAb 19E10, 1 µg/ml) processes do not overlap with dendritic MAP-2 (I: Sigma mouse mAb, 1:1000) staining (J: overlay). Scale bars, A–F:, 20 µm; D inset: 50 µm ; G: 10 µm; H–J: 10 µm.

Article Snippet: We also characterize the localization of exogenously expressed Slo1 α subunits in hippocampal neurons developing in culture that exhibit a prominent localization of Slo1 in axons and presynaptic terminals. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Anti-slo1 antibodies used in this study L6/60 mouse monoclonal IgG2a GST Fusion protein 690–1196 Mapped to within 690–891 Alomone APC-021, lot AN-04 Rabbit polyclonal GST Fusion protein 1098–1196 Chemicon AB5228, lot 22040170 Rabbit polyclonal GST Fusion protein 1098–1196 Anti-slo1 antibodies used in other studies Martin et al., 2004 .

Techniques: Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells

doi: 10.1073/pnas.1703081114

Figure Lengend Snippet: Anti-LRRC26 antibody pulls down SLO1 protein in parotid, lacrimal gland, and colon. (A) Confirmation of LRRC26 association with SLO1 protein in mouse parotid. (A1) Total membrane proteins from parotid wt, Slo1−/−, and Lrrc26−/− mice were blotted with anti-SLO1 Ab (L6/60, Antibodies, Inc.), identifying the SLO1 protein in wt and Lrrc26−/− mice, but not Slo1−/− mice. (A2) Proteins immunoprecipitated by the ProSci LRRC26 Ab were Western blotted, showing that LRRC26 is present in both wt and Slo1−/− mice, but not in the LRRC26 KO mice. (A3) Following immunoprecipitation of parotid membrane proteins with the LRRC26 Ab, SLO1 protein is identified in wt immunoprecipitated proteins, but not in Slo1−/− or Lrrc26−/− proteins. (A4) Aliquots of the parotid membrane protein preparations were blotted with a Na/K ATPase1A1 Ab to confirm that similar amounts of proteins were applied in all cases. (B1) Lacrimal gland total membrane proteins were blotted with the anti-SLO1 Ab. (B2) wt and Slo1−/−, but not Lrrc26−/−, lacrimal gland proteins contain LRRC26 protein. (B3) SLO1 protein is found in lacrimal gland membrane proteins immunoprecipitated with the LRRC26 Ab. (B4) Aliquots of lacrimal gland membrane proteins were blotted for ATP1A1. Note the markedly lower amounts of ATP1A1 in lacrimal gland, compared with parotid. (C1–C4) Slo1 protein in colon is also immunoprecipitated with the LRRC26 Ab.

Article Snippet: The coassembly of LRRC26 with SLO1 in parotid gland, lacrimal gland, and colon supports the view that LRRC26 is a BK regulatory subunit in these tissues and predicts that BK gating will be shifted leftward in these cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3. caption a7 Anti-LRRC26 antibody pulls down SLO1 protein in parotid, lacrimal gland, and colon. ( A ) Confirmation of LRRC26 association with SLO1 protein in mouse parotid. ( A1 ) Total membrane proteins from parotid wt , Slo1 −/− , and Lrrc26 −/− mice were blotted with anti-SLO1 Ab (L6/60, Antibodies, Inc.), identifying the SLO1 protein in wt and Lrrc26 −/− mice, but not Slo1 −/− mice. ( A2 ) Proteins immunoprecipitated by the ProSci LRRC26 Ab were Western blotted, showing that LRRC26 is present in both wt and Slo1 −/− mice , but not in the LRRC26 KO mice. ( A3 ) Following immunoprecipitation of parotid membrane proteins with the LRRC26 Ab, SLO1 protein is identified in wt immunoprecipitated proteins, but not in Slo1 −/− or Lrrc26 −/− proteins. ( A4 ) Aliquots of the parotid membrane protein preparations were blotted with a Na/K ATPase1A1 Ab to confirm that similar amounts of proteins were applied in all cases. ( B1 ) Lacrimal gland total membrane proteins were blotted with the anti-SLO1 Ab. ( B2 ) wt and Slo1 −/− , but not Lrrc26 −/− , lacrimal gland proteins contain LRRC26 protein. ( B3 ) SLO1 protein is found in lacrimal gland membrane proteins immunoprecipitated with the LRRC26 Ab. ( B4 ) Aliquots of lacrimal gland membrane proteins were blotted for ATP1A1.

Techniques: Membrane, Immunoprecipitation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Knockout of the LRRC26 subunit reveals a primary role of LRRC26-containing BK channels in secretory epithelial cells

doi: 10.1073/pnas.1703081114

Figure Lengend Snippet: LRRC26 KO mimics effect of SLO1 KO in reducing K+ efflux in salivary gland secretions. (A) Potassium content was measured from pilocarpine-induced fluid secretion from in vivo parotid glands, for wt (gray) and LRRC26 KO animals. Height of bars shows mean with error bars indicating SEM, whereas circles show individual determinations. Each determination is the average of secretion measured separately from both glands in a single animal, except in one case where only a single gland was obtained. (B) Two bars on the left compare potassium content in submandibular gland saliva from an in vivo measurement, and the two bars on the right show ex vivo potassium content of submandibular salivary secretion. For all comparisons between wt and Lrrc26−/− glands in both A and B, P < 0.001 for the t test.

Article Snippet: The coassembly of LRRC26 with SLO1 in parotid gland, lacrimal gland, and colon supports the view that LRRC26 is a BK regulatory subunit in these tissues and predicts that BK gating will be shifted leftward in these cells. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3. caption a7 Anti-LRRC26 antibody pulls down SLO1 protein in parotid, lacrimal gland, and colon. ( A ) Confirmation of LRRC26 association with SLO1 protein in mouse parotid. ( A1 ) Total membrane proteins from parotid wt , Slo1 −/− , and Lrrc26 −/− mice were blotted with anti-SLO1 Ab (L6/60, Antibodies, Inc.), identifying the SLO1 protein in wt and Lrrc26 −/− mice, but not Slo1 −/− mice. ( A2 ) Proteins immunoprecipitated by the ProSci LRRC26 Ab were Western blotted, showing that LRRC26 is present in both wt and Slo1 −/− mice , but not in the LRRC26 KO mice. ( A3 ) Following immunoprecipitation of parotid membrane proteins with the LRRC26 Ab, SLO1 protein is identified in wt immunoprecipitated proteins, but not in Slo1 −/− or Lrrc26 −/− proteins. ( A4 ) Aliquots of the parotid membrane protein preparations were blotted with a Na/K ATPase1A1 Ab to confirm that similar amounts of proteins were applied in all cases. ( B1 ) Lacrimal gland total membrane proteins were blotted with the anti-SLO1 Ab. ( B2 ) wt and Slo1 −/− , but not Lrrc26 −/− , lacrimal gland proteins contain LRRC26 protein. ( B3 ) SLO1 protein is found in lacrimal gland membrane proteins immunoprecipitated with the LRRC26 Ab. ( B4 ) Aliquots of lacrimal gland membrane proteins were blotted for ATP1A1.

Techniques: In Vivo, Ex Vivo