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: 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: Oxidative Medicine and Cellular Longevity

Article Title: Nicorandil Attenuates LPS-Induced Acute Lung Injury by Pulmonary Endothelial Cell Protection via NF- κ B and MAPK Pathways

doi: 10.1155/2019/4957646

Figure Lengend Snippet: Nico ameliorated LPS-induced ALI and inflammation. (a) Nico increased LPS-induced Kir6.1 and Kir6.2 downregulation in the lung. (b, c) Lung sections stained with H&E showed severe injury in the LPS group which was attenuated by Nico pretreatment. The data revealed a high score for the LPS-treated group which was decreased in the Nico-pretreated group. (d) Nico pretreatment significantly reduced LPS-induced protein leakage in BALF. (e, f) Nico alleviated LPS-induced increments of MPO activities in BALF and lung homogenate. (g, h) Nico prevented the production of TNF- α and IL-1 β in lung homogenate. Data were shown as mean ± SEM ( n = 6 − 8). Statistically significant differences: ∗ P < 0.05 versus the control group; # P < 0.05 versus the LPS group. Scale bar: 50 μ m.

Article Snippet: Then, the transferred membranes were incubated with primary antibodies against Kir6.1 (Alomone Labs, Jerusalem, Israel), Kir6.2 (Abcam), NF- κ B p-p65/p65, p-i κ B- α /i κ B- α , p-p38/p38, p-ERK/ERK, p-JNK/JNK, intercellular adhesion molecule-1 (ICAM-1), cleaved-caspase-3 (c-caspase-3), caspase-9 (1 : 1000, Cell Signaling Technology), endothelial nitric oxide synthase (eNOS) (1 : 1000, Santa Cruz), inducible nitric oxide synthase (iNOS) (1 : 1000, Millipore), CCAAT/enhancer-binding protein homologous protein (CHOP), vascular cell adhesion molecule-1 (VCAM-1), VE-cadherin, Nox4 (1 : 1000), MnSOD (1 : 5000, Abcam), and β -actin (1 : 5000, Proteintech, Rosemont, USA) overnight.

Techniques: Staining

Journal: PLoS ONE

Article Title: Unidirectional Photoreceptor-to-Müller Glia Coupling and Unique K + Channel Expression in Caiman Retina

doi: 10.1371/journal.pone.0097155

Figure Lengend Snippet: ( A ) Inwardly rectifying potassium channels Kir4.1 (green) are localized in the area of the photoreceptors and in outer nuclear layer (red arrowhead), but not in Müller cell processes such as stalks or endfeet. This is different from most of vertebrates where Kir4.1 channels were found in Müller cells . ( B ) Glial Müller cell specific marker vimentin, V (green); photoreceptor specific alpha-1 subunit of transducin, Gαt1 (red); nucleus-specific marker, DAPI (blue). Vimentin staining is observed in endfeet, somata, stalks and in distal processes (yellow arrowhead points somata, white arrow shows stalk). DAPI staining (blue) shows nuclei of retinal cells. Alpha-1 subunit of transducin (Gαt1, red) is a marker of the second messenger G-protein cascade and it is found mostly in photoreceptors. ( C ) ATP-dependent K + channel, Kir6.1 (black), is localized in stalks (white arrow) and in endfeet, while ( D ) two pore domain acid sensitive K + channels, TASK-1 (black) are found in whole Müller cell compartments: in endfeet (black arrowhead), stalks (white arrow), somata (yellow arrowhead) and in distal processes (white arrowhead). Scale bar = 20 µm. ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, ONL-outer nuclear layer, IS- inner segments of photoreceptors.

Article Snippet: We used the following primary antibodies: (i) for glial markers: mouse anti-S100β (1∶1000; Sigma-Aldrich, S2532), mouse anti-glial fibrillary acidic protein (GFAP; 1∶300; Sigma-Aldrich, G6171), mouse anti-glutamine synthetase (GS; 1∶500; Millipore, MAB302), mouse anti-vimentin (1∶300; PROGEN Biotechnik GmbH, Heidelberg, Germany, VIM 3B4), (ii) for channel proteins: rabbit anti-TASK-1 (1∶200; Santa Cruz Biotechnology, SC-28635), rabbit anti TREK-2 (1∶500; Alomone), rabbit anti TASK-3 (1∶300; Alomone), mouse anti-Kir4.1 (1∶300; Sigma-Aldrich, WH0003766M1), rabbit anti-Kir4.1 (1∶200; Alomone, APC-035), rabbit anti-Kir6.1 (1∶200; Alomone, APC-105), rabbit anti-Kir6.1, -Kir6.2, and -SUR1 (1∶200; 1∶150; 1∶200 respectively; R.W.

Techniques: Marker, Staining

Journal: PLoS ONE

Article Title: Unidirectional Photoreceptor-to-Müller Glia Coupling and Unique K + Channel Expression in Caiman Retina

doi: 10.1371/journal.pone.0097155

Figure Lengend Snippet: ( A ) Inwardly rectifying potassium channels Kir4.1 (green) are localized in the area of the photoreceptors and in outer nuclear layer (red arrowhead), but not in Müller cell processes such as stalks or endfeet. This is different from most of vertebrates where Kir4.1 channels were found in Müller cells . ( B ) Glial Müller cell specific marker vimentin, V (green); photoreceptor specific alpha-1 subunit of transducin, Gαt1 (red); nucleus-specific marker, DAPI (blue). Vimentin staining is observed in endfeet, somata, stalks and in distal processes (yellow arrowhead points somata, white arrow shows stalk). DAPI staining (blue) shows nuclei of retinal cells. Alpha-1 subunit of transducin (Gαt1, red) is a marker of the second messenger G-protein cascade and it is found mostly in photoreceptors. ( C ) ATP-dependent K + channel, Kir6.1 (black), is localized in stalks (white arrow) and in endfeet, while ( D ) two pore domain acid sensitive K + channels, TASK-1 (black) are found in whole Müller cell compartments: in endfeet (black arrowhead), stalks (white arrow), somata (yellow arrowhead) and in distal processes (white arrowhead). Scale bar = 20 µm. ILM-inner limiting membrane, GCL-ganglion cell layer, IPL-inner plexiform layer, INL-inner nuclear layer, ONL-outer nuclear layer, IS- inner segments of photoreceptors.

Article Snippet: We used the following primary antibodies: (i) for glial markers: mouse anti-S100β (1∶1000; Sigma-Aldrich, S2532), mouse anti-glial fibrillary acidic protein (GFAP; 1∶300; Sigma-Aldrich, G6171), mouse anti-glutamine synthetase (GS; 1∶500; Millipore, MAB302), mouse anti-vimentin (1∶300; PROGEN Biotechnik GmbH, Heidelberg, Germany, VIM 3B4), (ii) for channel proteins: rabbit anti-TASK-1 (1∶200; Santa Cruz Biotechnology, SC-28635), rabbit anti TREK-2 (1∶500; Alomone), rabbit anti TASK-3 (1∶300; Alomone), mouse anti-Kir4.1 (1∶300; Sigma-Aldrich, WH0003766M1), rabbit anti-Kir4.1 (1∶200; Alomone, APC-035), rabbit anti-Kir6.1 (1∶200; Alomone, APC-105), rabbit anti-Kir6.1, -Kir6.2, and -SUR1 (1∶200; 1∶150; 1∶200 respectively; R.W.

Techniques: Marker, Staining

Journal: Journal of Neuroinflammation

Article Title: Oral administration of the K ATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

doi: 10.1186/1742-2094-8-149

Figure Lengend Snippet: Western blotting show expression of both Kir6.1 and Kir6.2 K ATP channel pore-forming subunits in unstimulated and LPS/IFNγ-stimulated BV-2 cells (A, left) and microglial primary cultures (A, Right). Staining for the microglial cell membrane marker CD11b (B and E) and the K ATP channel subunits Kir 6.1 (C) or Kir 6.2 (F) showed colocalization in BV-2 microglia, indicating the expression of the K ATP channel at the cytoplasmic membrane (D and G, white arrows) . Control: unstimulated cells; L+I: cells stimulated with LPS and IFNγ. Scale bar = 30 μm.

Article Snippet: After washing in Tris-buffered saline (TBS: 20 mM Tris, 0.15 M NaCl, pH 7.5) for 5 min, dipping in methanol for 10 s and drying in air, the membranes were incubated with the following primary antibodies overnight at 4°C: polyclonal rabbit anti-Kir 6.1 or polyclonal rabbit anti-Kir 6.2 (both 1:500, Alomone, Jerusalem, Israel), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS) (1:200, Millipore, Bedford, MA, USA), polyclonal rabbit anti-cyclooxygenase-2 (1:2000, Santa Cruz Biotechnology, St. Cruz, CA, USA) and monoclonal mouse anti-β-actin (1:50000, Sigma-Aldrich, St. Louis, MO, USA) diluted in immunoblot buffer (TBS containing 0.05% Tween-20 and 5% non-fat dry milk).

Techniques: Western Blot, Expressing, Staining, Marker

Journal: Journal of Neuroinflammation

Article Title: Oral administration of the K ATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

doi: 10.1186/1742-2094-8-149

Figure Lengend Snippet: Western blotting show expression of both Kir6.1 and Kir6.2 K ATP channel pore-forming subunits in unstimulated and LPS/IFNγ-stimulated BV-2 cells (A, left) and microglial primary cultures (A, Right). Staining for the microglial cell membrane marker CD11b (B and E) and the K ATP channel subunits Kir 6.1 (C) or Kir 6.2 (F) showed colocalization in BV-2 microglia, indicating the expression of the K ATP channel at the cytoplasmic membrane (D and G, white arrows) . Control: unstimulated cells; L+I: cells stimulated with LPS and IFNγ. Scale bar = 30 μm.

Article Snippet: Cells were then incubated with primary antibodies anti-Kir6.1 and anti-Kir6.2 (1:300 dilution, Alomone, Jerusalem, Israel), anti-CD11b (1:500 dilution, Serotec, Oxford, England, UK) at 4°C overnight, followed by secondary antibodies Alexa ® 488 and 596 (1:500, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h in blocking solution.

Techniques: Western Blot, Expressing, Staining, Marker

Journal: Journal of Neuroinflammation

Article Title: Oral administration of the K ATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

doi: 10.1186/1742-2094-8-149

Figure Lengend Snippet: Confocal double immunofluorescence images of CD11b (red, A and D) and Kir6.2 (green, B and E) in spinal cord sections from healthy control mice (Ctrl) or MOG 35-55 EAE mice . A slight intensity was found for Kir6.2 in healthy section showing low localization of the K ATP Kir6.2 subunit in CD11b-positive cells (white arrows, C). However, higher intensity of Kir6.2 subunit in CD11b reactive cells showing a strong colocalization of both (white arrows, F) was observed. Western blotting for Kir6.2 in total protein homogenates from lumbar-sacral and thoracic-cervical regions of the spinal cord from non-immunized control animals (control, G) and EAE mice (EAE, G) show an increase in Kir6.2 expression in EAE mice. This increase is statistically significant in the thoracic-cervical level of the spinal cord (H). Results are shown as mean ± SEM. ** p < 0.01 between control and EAE. Scale bar = 30 μm.

Article Snippet: Cells were then incubated with primary antibodies anti-Kir6.1 and anti-Kir6.2 (1:300 dilution, Alomone, Jerusalem, Israel), anti-CD11b (1:500 dilution, Serotec, Oxford, England, UK) at 4°C overnight, followed by secondary antibodies Alexa ® 488 and 596 (1:500, Molecular Probes, Invitrogen, Eugene, OR, USA) for 1 h in blocking solution.

Techniques: Immunofluorescence, Western Blot, Expressing