Journal: Molecular Pain

Article Title: K ATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

doi: 10.1186/1744-8069-6-6

Figure Lengend Snippet: Expression of K ATP channel subunits in DRG at the mRNA and protein level . A . RT-PCR product bands consistent with the presence of mRNA encoding K ATP channel subunits in DRG from control rats . Total mRNA was isolated from the L5 DRG. Amplified products appeared at positions corresponding to the expected base pair lengths of 168 (Kir6.2), 182 (SUR1), 110 (Kir6.1), 124 (SUR2) and 315 (18S). B . Western blotting using antibodies against K ATP channel subunits in DRG of control rats . Kir6.2 antibody recognized one immunoreactive band at 37 kDa. SUR1 antibody revealed prominent immunoreactive bands at 150 and 37 kDa, and a less pronounced band at 125 kDa. The band at 150 kDa indicates glycosylated SUR1 bound to Kir6.2. Kir6.1 did not detect any band in DRG tissue, while a band of 37 kDa was detected in samples of brain tissue. Two immunoreactive bands at 150 and 74 kDa were detected by SUR2 antibody. Molecular size markers are shown on the left.

Article Snippet: After blocking with 4% normal goat serum for 1 h at room temperature, slides were incubated in polyclonal rabbit antibodies to Kir6.1, Kir6.2 (1:500, Alomone, Jerusalem, Israel), and SUR1 or SUR2 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Western Blot

Journal: Molecular Pain

Article Title: K ATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

doi: 10.1186/1744-8069-6-6

Figure Lengend Snippet: Presence and distribution of Kir6.2, SUR1, and SUR2 subunits in DRG neurons and satellite glial cells from control rats . Samples of DRG slices were co-labeled with DAPI, which stains nuclei (blue), and the antibody against each individual subunit (red) (A-D). A . Kir6.1 immunofluorescence was absent in DRG. In contrast the same antibody revealed immunostaining in positive controls (rat brain and aorta smooth muscle; not shown). B . Immunofluorescence against Kir6.2 is identified on plasma membranes (yellow arrowheads) and cytosol (white arrow). Most satellite glial cells also stained positive for Kir6.2. C . Immunofluorescence against SUR1 is observed in the plasma (yellow arrowheads) and nuclear membranes (purple color), as well as along the axons (single yellow arrowhead). Satellite glial cells also stained positive. D . Staining against the SUR2 subunit is observed in the plasma membrane (yellow arrowheads), nuclear membrane (purple color), and the cytosol (white arrow). Satellite glial cells also stained positive. In order to confirm the localization of staining in the plasmalemmal membrane of neurons versus the satellite cell membrane, we examined dissociated DRG cells, stained with the same antibodies, using confocal microscopy. These images clearly showed that neuronal plasmalemmal membrane stained positive for SUR1 ( E ), Kir6.2 ( F ), and SUR2 ( G ). Nuclear envelops also stained positive ( E and G , single yellow arrowhead). Distinct positive staining was also observed in satellite cells ( G ). In E , images correspond to 5 sequential confocal images of z-projections (with spacing increments of 1 μm).

Article Snippet: After blocking with 4% normal goat serum for 1 h at room temperature, slides were incubated in polyclonal rabbit antibodies to Kir6.1, Kir6.2 (1:500, Alomone, Jerusalem, Israel), and SUR1 or SUR2 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Labeling, Immunofluorescence, Immunostaining, Staining, Confocal Microscopy

Journal: Molecular Pain

Article Title: K ATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

doi: 10.1186/1744-8069-6-6

Figure Lengend Snippet: Preincubation with anti-SUR1 antibody abolishes the blocking effect of glybenclamide on single K ATP channel opening in excised membrane patches . Neurons preincubated with anti-SUR1 antibody (n = 5, D, E, F ; purple) were compared to neurons preincubated in antibody-free solution (n = 7, A, B, C ). Horizontal arrows indicate closed channel. A . Representative trace of K ATP channel activity in patch excised from a neuron preincubated in antibody-free solution. In these neurons glybenclamide inhibited channel activity in a concentration-dependent fashion. B . Marked channel activity occurred upon excision of patch (vertical arrow in A ) into an ATP-free solution. C . Glybenclamide 1000 nM blocked channel activity under control conditions. D . Representative trace of K ATP channel activity in patch excised from a neuron preincubated with anti-SUR1 antibody. E . Excision of patch (vertical arrow in D ) into an ATP-free external solution also activated channel. Cell-free patch exhibited similar K ATP single channel activity as in controls ( B ). F . In contrast to neurons preincubated in antibody-free solution ( C ), glybenclamide 1000 nM failed to block channel activity after preincubation with anti-SUR1 antibody. G . Blocking effect of glybenclamide under control conditions is shown in the concentration-response curve (dotted-line; lower trace). Cumulative application of glybenclamide failed to block channel activity after preincubation with anti-SUR1 antibody, as indicated by the less steep concentration-response curve in G (solid line; upper trace). Means ± SD are shown. *:p < 0.05 versus glybenclamide 1 nM; §: p < 0.05 versus control. (Student's t tests were used for intergroup, and Bonferroni tests for intragroup post hoc comparisons).

Article Snippet: After blocking with 4% normal goat serum for 1 h at room temperature, slides were incubated in polyclonal rabbit antibodies to Kir6.1, Kir6.2 (1:500, Alomone, Jerusalem, Israel), and SUR1 or SUR2 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Blocking Assay, Activity Assay, Concentration Assay

Journal: Molecular Pain

Article Title: K ATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

doi: 10.1186/1744-8069-6-6

Figure Lengend Snippet: Colocalization studies in DRG neurons . A-C. Colocalization of BODIPY-Glybenclamide staining of SUR1 subunits ( A ), with anti-Kir6.2 antibody ( B ), showing that SUR1 subunits are co-expressed with Kir6.2 subunits in the same complexes ( C : merged). D-F . Co-localization of anti-SUR1 antibody ( D ) with anti-CGRP antibody ( E ) in DRG studied under fluorescent microscopy. Merged image ( F ) shows anti-SUR1 immunofluorescence present in small, CGRP positive neurons, as well as in CGRP negative neurons. G - I . Co-localization of anti-SUR1 antibody ( G ) with anti-CGRP antibody ( H ) in dissociated neurons studied under confocal microscopy. Merged image ( I ) shows SUR1 immunofluorescence present in small, CGRP + neurons (arrows), as well as in CGRP negative neurons. K - M . Co-localization of anti-SUR1 antibody ( K ) with anti-Caspr antibody ( L ) in DRG studied under confocal microscopy. Merged image ( M ) shows that SUR1 immunofluorescence co-localizes with anti-Caspr staining in paranodal sites. Yellow arrowheads point to SUR1 positive SLI (in K ). N - P . Colocalization of anti-Kir6.2 antibody ( N ) with anti-Caspr antibody ( O ) in DRG studied under confocal microscopy. Merged image ( P ) shows that anti-SUR1 immunofluorescence colocalizes with anti-Caspr staining in paranodal sites, adjacent to Ranvier's nodes (White arrows point at paranodal K ATP channels). This colocalization of Caspr with SUR1 or Kir6.2 indicates that K ATP channels of the Kir6.2/SUR1 subtype are present in paranodal sites (white arrows) adjacent to nodes of Ranvier. All samples are from slices within DRG tissue.

Article Snippet: After blocking with 4% normal goat serum for 1 h at room temperature, slides were incubated in polyclonal rabbit antibodies to Kir6.1, Kir6.2 (1:500, Alomone, Jerusalem, Israel), and SUR1 or SUR2 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Staining, Microscopy, Immunofluorescence, Confocal Microscopy

Journal: Molecular Pain

Article Title: K ATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

doi: 10.1186/1744-8069-6-6

Figure Lengend Snippet: Distribution of K ATP subunits on DRG examined by electron microscopy . Samples from slices within DRG tissue were treated with antibodies against Kir6.2 or SUR1, which were labeled with gold particles (shown as black dots). A . Anti-Kir6.2 staining on axonal membrane (red arrow) and myelin sheath (yellow arrowhead). B . Anti-SUR1 staining on axonal membrane (red arrows). C . Anti-SUR1 staining on axonal membrane (red arrows) and into a SLI (yellow arrowheads). The axons are marked by red asterisks.

Article Snippet: After blocking with 4% normal goat serum for 1 h at room temperature, slides were incubated in polyclonal rabbit antibodies to Kir6.1, Kir6.2 (1:500, Alomone, Jerusalem, Israel), and SUR1 or SUR2 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Electron Microscopy, Labeling, Staining

Journal: Molecular Pain

Article Title: K ATP channel subunits in rat dorsal root ganglia: alterations by painful axotomy

doi: 10.1186/1744-8069-6-6

Figure Lengend Snippet: Distribution of anti-SUR1 immunofluorescence in the subpopulations of NF200+ and NF200- neurons in control (A-D) and SNL (E-H) DRG . A, E: Red anti-SUR1 immunofluorescence is observed on plasma and nuclear membranes, satellite glial cells, and along the peripheral nerve fibers. White arrows point to SLI. These are more intense and funnel shaped in controls ( A ) compared to SNL ( E ), wherein SLI loose their characteristic funnel shape, and appear less intense, thin and disorganized. B , F . Anti-NF 200 staining (green), distinguishes two DRG neuronal subpopulations, corresponding to larger, myelinated NF200+ and to smaller, non-myelinated NF200- fibers. C , G . Merged images showing the difference in distribution of SUR1 staining in each neuronal subgroup between SS and SNL DRG. SLI are shown with arrows in C . D , H : Bright field images. SLI are shown by yellow arrowheads in D . I ( 1-4 ). Bargraphs showing the differences in the prevalence of SUR1+ staining between control (C) and SNL NF200+ ( I1 ) and between control (C) and SNL NF200- neuronal somata ( I2 ), compared by Fisher's exact tests. I3 . Bargraphs showing the decreased prevalence of SUR1+ SLI between control (C) and SNL axons, compared by Fisher's exact test. I4 . Scatterplots showing the difference in the intensity of SUR1 immunofluorescence in SLI between axons from SNL versus control DRG. Arbitrary fluorescence units are used. Comparisons were made by Student's t test.

Article Snippet: After blocking with 4% normal goat serum for 1 h at room temperature, slides were incubated in polyclonal rabbit antibodies to Kir6.1, Kir6.2 (1:500, Alomone, Jerusalem, Israel), and SUR1 or SUR2 (1:50, Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

doi: 10.1074/jbc.M110.147868

Figure Lengend Snippet: Decreased KATP channel subunit expression in ankyrin-B+/− hearts. Detergent-soluble ankyrin-B+/− heart lysates expressed significantly less ankyrin-B (A), Kir6.2 (B), SUR1 (C), SUR2A (D), Na/K-ATPase (H), and NCX1 (I). There were no significant differences in the expression of ankyrin-G (E), Kir6.1 (F), or NHERF1 (loading control; G). J, quantitative data demonstrating a significant decrease in the expression of ankyrin-B, NCX1, Na/K-ATPase, and KATP channel proteins in ankyrin-B+/− compared with wild-type detergent-soluble mouse heart lysates. Expression of proteins was normalized to wild-type expression. IB, immunoblot. *, p < 0.05.

Article Snippet: Antibodies The following primary antibodies were used for immunoblotting protocols and/or immunofluorescent staining: anti-ankyrin-B (polyclonal and monoclonal), anti-ankyrin-G (polyclonal), anti-Kir6.2 and anti-Kir6.1 (Alomone), anti-SUR1 and anti-SUR2A (Santa Cruz Biotechnology), anti-NHERF1 (polyclonal), anti-NCX1 (RDI), anti-Na/K-ATPase (Upstate), anti-SERCA2 (polyclonal), anti-Ca v 1.2 (polyclonal), and anti-Na v 1.5 (polyclonal).

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

doi: 10.1074/jbc.M110.147868

Figure Lengend Snippet: Ankyrin-B, Kir6.2, and SUR1/SUR2A associate in a ternary complex. A and B, co-immunoprecipitation experiments demonstrated association of ankyrin-B, Kir6.2, and SUR1/SUR2A. HEK293 cells (which express ankyrin-B, but not Kir6.2, SUR1, or SUR2A; C, first lane) were transfected with Kir6.2 and/or SUR1/SUR2A as indicated. Cell lysates were immunoprecipitated (IP) with either anti-Kir6.2 Ig (A) or anti-ankyrin-B Ig (B), and bound protein was analyzed by SDS-PAGE and immunoblotting (IB) using anti-ankyrin-B, anti-Kir6.2, anti-SUR1, and anti-SUR2A antibodies. Note that Kir6.2 expression was required for ankyrin-B/SUR1 and ankyrin-B/SUR2A association. C, expression control demonstrating that HEK293 cells express transfected proteins.

Article Snippet: Antibodies The following primary antibodies were used for immunoblotting protocols and/or immunofluorescent staining: anti-ankyrin-B (polyclonal and monoclonal), anti-ankyrin-G (polyclonal), anti-Kir6.2 and anti-Kir6.1 (Alomone), anti-SUR1 and anti-SUR2A (Santa Cruz Biotechnology), anti-NHERF1 (polyclonal), anti-NCX1 (RDI), anti-Na/K-ATPase (Upstate), anti-SERCA2 (polyclonal), anti-Ca v 1.2 (polyclonal), and anti-Na v 1.5 (polyclonal).

Techniques: Immunoprecipitation, Transfection, SDS Page, Western Blot, Expressing