Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Antibodies Used for Immunohistochemistry

Article Snippet: The results obtained showed that only the Kir6.1 antibody was detecting the HEK293T-cells lysate, as is shown in . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue.

Techniques:

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Antibody validation. (A) Upper images show the result of a western blot analysis of human Kir6.2 in five specimens of human brain tissue (lanes 1 to 5: 2, 4 corresponding to contusion specimens and 1, 3, 5 corresponding to healthy tissue) using two independent antibodies (Alomone Ab and GeneTex Ab). The bar graphs are shown as a visual aid for the inmunoblot results. Optical density with both antibodies was compared and resulted in a Pearson correlation coefficient of 0.88 (p = 0.049). The scatterplot is shown as a visual aid to the immunoblot results. (B) Fluorescence labeling of Kir6.2 using Alomone Ab (red) and SantaCruz Ab (green); merged images in yellow. (C) Western blot analysis of a human recombinant Kir6.2 protein (ab114436; Abcam, Cambridge, UK) (1) tagged with green fluorescent protein (GFP) and GFP recombinant protein (2) used as a negative control. (D) Kir6.2 expression (green) in a cell line of human cardiomyocytes (P10451; Innoprot, Derio, Spain) used as a positive control. Nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI).

Article Snippet: The results obtained showed that only the Kir6.1 antibody was detecting the HEK293T-cells lysate, as is shown in . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue.

Techniques: Western Blot, Fluorescence, Labeling, Recombinant, Negative Control, Expressing, Positive Control

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue. Kir6.1 shows labeling in blood vessel-like structures and Kir6.2 in neuronal-like structures. This shows that the Kir6.2 antibody does not cross-react with the Kir6.1 epitope. (B) Western blot analysis of a lysate of HEK293T-cells transfected with an empty vector (lane 1) and HEK293T cells that overexpress Kir6.1 (NBL1-12174; Novus Biological, Littleton, CO) (lane 2). The membrane shown on the left was incubated with an anti-Kir6.1 antibody and the one on the right with an anti-Kir6.2 antibody. Only the Kir6.1 antibody was detecting the HEK293T-cells lysate showing that cross-reactivity does not occur between the Kir6.2 antibody and the Kir6.1 epitope.

Article Snippet: The results obtained showed that only the Kir6.1 antibody was detecting the HEK293T-cells lysate, as is shown in . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue.

Techniques: Staining, Labeling, Western Blot, Transfection, Plasmid Preparation, Incubation

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Kir6.2 protein levels in control and contusion samples. (A) Box plots of Kir6.2 relative intensity in control and contusion samples, showing a significant increase of Kir6.2 expression (p = 0.014) in contusion specimens; data normalized to β-actin loading controls. (B) Representative image of a western blot showing a clear difference between control and contusion samples.

Article Snippet: The results obtained showed that only the Kir6.1 antibody was detecting the HEK293T-cells lysate, as is shown in . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue.

Techniques: Expressing, Western Blot

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Expression of Kir6.2 in different cell types. The first three columns of the montage show Kir6.2 expression in different cell types of the peri-contusional tissue and the last three show Kir6.2 expression in control tissue. Peri-contusional tissue: fluorescent double labeling for NeuN/ glial fibrillary acidic protein (GFAP)/Iba1/CD31 (green) and Kir6.2 (red). Merged images are presented in the third column. Control tissue: fluorescent double labeling for NeuN/GFAP/Iba1/CD31 (green) and Kir6.2 (red). Merged images are presented in the last column. In controls, the Kir6.2 expression was evident in neurons and microglia but was minimal in astrocytes (GFAP-positive cells). Original magnification = 100x. Scale: 20 μm. Nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI).

Article Snippet: The results obtained showed that only the Kir6.1 antibody was detecting the HEK293T-cells lysate, as is shown in . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue.

Techniques: Expressing, Labeling

Journal: Journal of Neurotrauma

Article Title: Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K + Channels, Is Overexpressed in Human Posttraumatic Brain Contusions

doi: 10.1089/neu.2017.5619

Figure Lengend Snippet: Kir6.2 expression in neurons, glial cells, and microglia. From left to right, representative box plots of Kir6.2 expression in neurons (percentage), in glial cells, and microglia (semi-quantitative scale from 0 to 3). The results show a significantly increased expression of Kir6.2 in glial cells from contusion samples (Wilcoxon rank sum test; p = 0.03), but non-significant differences in neurons and microglia. Asterisks indicate statistically significant differences (*p ≤ 0.05).

Article Snippet: The results obtained showed that only the Kir6.1 antibody was detecting the HEK293T-cells lysate, as is shown in . fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 2. caption a7 Cross-reactivity Kir6.2-Kir6.1. (A) Staining of Kir6.1 (green; APC-105; Alomone Labs) and Kir6.2 (red; APC-020; Alomone Labs) in the same section of mouse brain tissue.

Techniques: Expressing

Journal:

Article Title: Sex differences in the contribution of ATP-sensitive K + channels in trigeminal ganglia under an acute muscle pain condition

doi: 10.1016/j.neuroscience.2011.01.045

Figure Lengend Snippet: Conventional RT-PCR analysis demonstrated the presence of mRNAs for Kir6.1, Kir6.2, SUR1, and SUR2 subunits in the TG of both male and female rats.

Article Snippet: Four KATP subunit antibodies were used. (1) A polyclonal rabbit antibody corresponding to amino acid residues 382–396 of rat Kir6.1 was raised against the peptide C-KRNSMRRNNSMRRSN (1:500, Alomone Labs). (2) A polyclonal rabbit antibody corresponding to amino acid residues 372–385 of rat Kir6.2 was raised against the peptide C-SVAVAKAKPKFSIS (1:500, Alomone Labs). (3) A polyclonal goat antibody was raised against a peptide mapping the c-terminus of human SUR1 (1:200, Santa Cruz Biotechnology). (4) A polyclonal goat antibody was raised against amino acids 921–1000 mapping an internal region of human SUR2 (1:200, Santa Cruz Biotechnology).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Sex differences in the contribution of ATP-sensitive K + channels in trigeminal ganglia under an acute muscle pain condition

doi: 10.1016/j.neuroscience.2011.01.045

Figure Lengend Snippet: Western blot experiments confirmed that protein for each KATP subunit is expressed in TG. (Top) Examples of immunoblots for Kir and SUR subunits along with GAPDH from TG of males and females in Pro and Di phases are shown. (Bottom) The group data showed that Kir6.2 protein level in female TG was substantially less compared to that of male. Other KATP subunits were expressed at comparable levels between the two sexes (n=6 for each group). p<0.05

Article Snippet: Four KATP subunit antibodies were used. (1) A polyclonal rabbit antibody corresponding to amino acid residues 382–396 of rat Kir6.1 was raised against the peptide C-KRNSMRRNNSMRRSN (1:500, Alomone Labs). (2) A polyclonal rabbit antibody corresponding to amino acid residues 372–385 of rat Kir6.2 was raised against the peptide C-SVAVAKAKPKFSIS (1:500, Alomone Labs). (3) A polyclonal goat antibody was raised against a peptide mapping the c-terminus of human SUR1 (1:200, Santa Cruz Biotechnology). (4) A polyclonal goat antibody was raised against amino acids 921–1000 mapping an internal region of human SUR2 (1:200, Santa Cruz Biotechnology).

Techniques: Western Blot

Journal:

Article Title: Sex differences in the contribution of ATP-sensitive K + channels in trigeminal ganglia under an acute muscle pain condition

doi: 10.1016/j.neuroscience.2011.01.045

Figure Lengend Snippet: Kir6.1 and Kir6.2 are expressed in trigeminal ganglion neurons (A and D, respectively). The somata of masseter afferents labeled by retrograde transport of Fast Blue (FB; B and E) expressed Kir6.1 (C) or Kir6.2 (F). Bar graphs represent sex differences in the percentages of Kir6.1 (top) and Kir6.2 (bottom) positive masseter afferents in TG. * p<0.05, **p<0.01 (n= 4 in each group). The estrus cycle phase of the female rats were not determined for these experiments.

Article Snippet: Four KATP subunit antibodies were used. (1) A polyclonal rabbit antibody corresponding to amino acid residues 382–396 of rat Kir6.1 was raised against the peptide C-KRNSMRRNNSMRRSN (1:500, Alomone Labs). (2) A polyclonal rabbit antibody corresponding to amino acid residues 372–385 of rat Kir6.2 was raised against the peptide C-SVAVAKAKPKFSIS (1:500, Alomone Labs). (3) A polyclonal goat antibody was raised against a peptide mapping the c-terminus of human SUR1 (1:200, Santa Cruz Biotechnology). (4) A polyclonal goat antibody was raised against amino acids 921–1000 mapping an internal region of human SUR2 (1:200, Santa Cruz Biotechnology).

Techniques: Labeling

Journal: iScience

Article Title: Kir6.2 -deficient mice develop somatosensory dysfunction and axonal loss in the peripheral nerves

doi: 10.1016/j.isci.2021.103609

Figure Lengend Snippet:

Article Snippet: Mouse: Kir6.2 -deficient , RIKEN BioResource Center , Cat# RBRC09393, RRID:IMSR_RBRC09393.

Techniques: Recombinant, Software

Journal:

Article Title: CARDIAC SULFONYLUREA RECEPTOR SHORT FORM-BASED CHANNELS CONFER A GLIBENCLAMIDE-INSENSITIVE KATP ACTIVITY

doi: 10.1016/j.yjmcc.2007.09.010

Figure Lengend Snippet: Design of new SUR2 antibodies and specificity tests. A: The 17-transmembrane helix model for SUR2 topology with positions for SUR2 antibodies shown. B: RT-PCR screening in COS1 cells contained a stably expressed KIR6.2 (top panel) or KIR6.1 (bottom panel). In each panel, Lane 1: PCR products amplified from a mouse cDNA library; Lane 2: Water control; Lanes 3−6: RT-PCR products amplified from selected candidates. Arrows indicate expected sizes of RT-PCR products. C: Western blot analysis to confirm COS1 lines stably expressing either a KIR6.2 (positive from Fig. 5B top panel, Lane 5) or a KIR6.1 (positive from Fig. 5B bottom panel, Lane 6). Arrows indicate detected sizes of Kir6.2 and Kir6.1 proteins. D-F: Specificity tests for T1, BNJ-2, BNJ-39 and BNJ-40 antibodies using isolated proteins from COS1 cells stably expressing a Kir6.2 pore and SUR2A, SUR2B or SUR1 cDNA. In all experiments, ∼25 μg of isolated protein isolated was loaded in each lane of a 4−12% MOPS NuPAGE gel. T1 (1: 2000), BNJ-2 (1:1000), BNJ-39 (1:2000) and BNJ-40 (1:1000) were used as primary antibodies. Secondary antibodies were added at 1:10000−1:12500. Arrows indicate detected protein sizes under our gel system and testing conditions.

Article Snippet: The mouse KIR6.2 [ 16 ] or KIR6.1 [ 17 , 18 ] gene was cloned from a mouse heart cDNA library (BD BioSciences, San Jose, CA) by PCR.

Techniques: Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Amplification, cDNA Library Assay, Western Blot, Expressing, Isolation

Journal:

Article Title: CARDIAC SULFONYLUREA RECEPTOR SHORT FORM-BASED CHANNELS CONFER A GLIBENCLAMIDE-INSENSITIVE KATP ACTIVITY

doi: 10.1016/j.yjmcc.2007.09.010

Figure Lengend Snippet: Co-IP results of the SUR2 short forms with Kir6.1 or Kir6.2. 5 μg of a specific antibody or control IgG was used to IP ∼100 μg purified membrane proteins isolated from the SUR2 mutant hearts. A: Control IgG, BNJ-39 and BNJ-40 were used in the forward IP experiments followed by a Western using anti-Kir6.2 (1:200). Anti-Kir6.2 was used in the reverse IP experiments followed by Westerns using either BNJ-39 (1:2000) or BNJ-40 (1:1000). B: Control IgG, BNJ-39 and BNJ-40 were used in the forward IP experiments followed by a Western using anti-Kir6.1 (1:200). Anti-Kir6.1 was used in the reverse IP experiment followed by a Western using BNJ-39 (1:2000). In all panels, secondary antibodies were added at 1:10000−1:12500. Arrows refer to detected protein sizes under our gel system and testing conditions. C: A schematic diagram of each deduced KATP complex based on results from this work. (I): 150/Kir6.1 or Kir6.2, (II) 150/28A/Kir6.1 or Kir6.2; (III): 68A/Kir6.1 or Kir6.2; (IV): 28B/Kir6.2.

Article Snippet: The mouse KIR6.2 [ 16 ] or KIR6.1 [ 17 , 18 ] gene was cloned from a mouse heart cDNA library (BD BioSciences, San Jose, CA) by PCR.

Techniques: Co-Immunoprecipitation Assay, Purification, Isolation, Mutagenesis, Western Blot