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: The Journal of Veterinary Medical Science

Article Title: ATP-dependent potassium channels contribute to motor regulation of esophageal striated muscle in rats

doi: 10.1292/jvms.19-0197

Figure Lengend Snippet: Expression of subunits of K ATP channels in the rat esophagus determined by RT-PCR. Homogenized samples from the rat tissues were used for RT-PCR. Amplified products of mRNA of Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B were detected in appropriate sizes ( n =3).

Article Snippet: The sections were then incubated with 2% normal goat serum at room temperature for 30 min. After rinsing in PBS, the sections were incubated with anti-SUR1 polyclonal antibody (catalog number: bs-3641R; lot number: 9B21M9; Bioss, Boston, MA, U.S.A.) diluted 1:100 or with anti-Kir6.1 polyclonal antibody (catalog number: bs-6468R; lot number: AF02227906; Bioss) diluted 1:100 at 4°C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: The Journal of Veterinary Medical Science

Article Title: ATP-dependent potassium channels contribute to motor regulation of esophageal striated muscle in rats

doi: 10.1292/jvms.19-0197

Figure Lengend Snippet: Histological examinations of the rat esophagus with paraffin section. (A) Kir6.1 immunoreactivity was observed mainly in the muscular layer in the rat esophagus. (B) The control staining using a normal rabbit IgG instead of antibody for K ATP channel. No specific staining was observed. L shows the lumen. Scale bar=100 µ m.

Article Snippet: The sections were then incubated with 2% normal goat serum at room temperature for 30 min. After rinsing in PBS, the sections were incubated with anti-SUR1 polyclonal antibody (catalog number: bs-3641R; lot number: 9B21M9; Bioss, Boston, MA, U.S.A.) diluted 1:100 or with anti-Kir6.1 polyclonal antibody (catalog number: bs-6468R; lot number: AF02227906; Bioss) diluted 1:100 at 4°C overnight.

Techniques: Paraffin Section, Staining

Journal: The Journal of Veterinary Medical Science

Article Title: ATP-dependent potassium channels contribute to motor regulation of esophageal striated muscle in rats

doi: 10.1292/jvms.19-0197

Figure Lengend Snippet: Histological examinations of the rat esophagus with cryostat section. The muscular layer of the rat esophagus showed immunoreactivity by antibodies not only for Kir6.1 (A) but also for SUR1 (B). (C) The control staining using a normal rabbit IgG instead of antibody for K ATP channel. No specific staining was observed. L shows the lumen. Scale bar=100 µ m.

Article Snippet: The sections were then incubated with 2% normal goat serum at room temperature for 30 min. After rinsing in PBS, the sections were incubated with anti-SUR1 polyclonal antibody (catalog number: bs-3641R; lot number: 9B21M9; Bioss, Boston, MA, U.S.A.) diluted 1:100 or with anti-Kir6.1 polyclonal antibody (catalog number: bs-6468R; lot number: AF02227906; Bioss) diluted 1:100 at 4°C overnight.

Techniques: Staining

Journal: Scientific Reports

Article Title: Ectopic overexpression of Kir6.1 in the mouse heart impacts on the life expectancy

doi: 10.1038/s41598-018-30175-5

Figure Lengend Snippet: Primer sets used for real-time PCR analyses.

Article Snippet: Kir6.1 transgenic mouse strains (Kir6.1TG line 634, 673, 105, and 111) used in this study are available through RIKEN BioResource Center (RBRC10018-21).

Techniques: Real-time Polymerase Chain Reaction

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