anti kir5 1 kcnj16 antibodies  (Alomone Labs)


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    Structured Review

    Alomone Labs anti kir5 1 kcnj16 antibodies
    Intracellular accumulation of polymyxin B in wild-type, KCNJ15 KO and <t>KCNJ16</t> KO HK-2 cells with the treatment of 25 µM polymyxin B and 50 µM BaCl 2 for 6 h. A Polymyxin B was immunostained with polymyxin <t>antibody</t> and visualized using Alexa Fluor-594 dye (red). The nucleus was counterstained with DAPI (blue). B The plots showing mean fluorescence intensities from each group. The value from control group has been deducted and the mean value from each replicate was plotted ( n = 4)
    Anti Kir5 1 Kcnj16 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kir5 1 kcnj16 antibodies/product/Alomone Labs
    Average 94 stars, based on 3 article reviews
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    anti kir5 1 kcnj16 antibodies - by Bioz Stars, 2022-11
    94/100 stars

    Images

    1) Product Images from "Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity"

    Article Title: Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-022-04316-z

    Intracellular accumulation of polymyxin B in wild-type, KCNJ15 KO and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B and 50 µM BaCl 2 for 6 h. A Polymyxin B was immunostained with polymyxin antibody and visualized using Alexa Fluor-594 dye (red). The nucleus was counterstained with DAPI (blue). B The plots showing mean fluorescence intensities from each group. The value from control group has been deducted and the mean value from each replicate was plotted ( n = 4)
    Figure Legend Snippet: Intracellular accumulation of polymyxin B in wild-type, KCNJ15 KO and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B and 50 µM BaCl 2 for 6 h. A Polymyxin B was immunostained with polymyxin antibody and visualized using Alexa Fluor-594 dye (red). The nucleus was counterstained with DAPI (blue). B The plots showing mean fluorescence intensities from each group. The value from control group has been deducted and the mean value from each replicate was plotted ( n = 4)

    Techniques Used: Fluorescence

    Polymyxin B induced significant electrophysiological changes and membrane depolarization in HK-2 cells. A The resting membrane potential in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 34, 18 and 10, respectively). B Input resistances in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 20, 18 and 10, respectively). C In current clamp mode, polymyxin B induced approximately 30 mV depolarization in WT cells and this was reversible. Depolarization was not induced in KCNJ15 KO cells. Polymyxin B induced membrane potential changes are shown aside ( n = 9, 10 and 7, respectively). D In voltage clamp mode, polymyxin B induced a statistically significant inward current (green) in wild-type HK-2 cells ( n = 8), but not in KCNJ15 KO cells ( n = 8). The current and reversal potential values are shown aside. E Fluorescent signal detection in HK-2 cells with DiBAC, 25 μM polymyxin B, and DiBAC plus 25 μM polymyxin B. F Proportions of DiBAC-positive in wild-type, KCNJ15 KO, and KCNJ16 KO HK-2 cells measured by flow cytometry. G Proportions of DiBAC-positive HK-2 cells in the control and BaCl 2 (10 μM) groups with or without 25 μM polymyxin B treatment measured by flow cytometry ( n = 5 for WT and n = 4 for KOs). Data are shown as box and whisker plots. One-way (for WT) or two-way (for KOs) ANOVA was employed for multi-group comparisons. ** p
    Figure Legend Snippet: Polymyxin B induced significant electrophysiological changes and membrane depolarization in HK-2 cells. A The resting membrane potential in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 34, 18 and 10, respectively). B Input resistances in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 20, 18 and 10, respectively). C In current clamp mode, polymyxin B induced approximately 30 mV depolarization in WT cells and this was reversible. Depolarization was not induced in KCNJ15 KO cells. Polymyxin B induced membrane potential changes are shown aside ( n = 9, 10 and 7, respectively). D In voltage clamp mode, polymyxin B induced a statistically significant inward current (green) in wild-type HK-2 cells ( n = 8), but not in KCNJ15 KO cells ( n = 8). The current and reversal potential values are shown aside. E Fluorescent signal detection in HK-2 cells with DiBAC, 25 μM polymyxin B, and DiBAC plus 25 μM polymyxin B. F Proportions of DiBAC-positive in wild-type, KCNJ15 KO, and KCNJ16 KO HK-2 cells measured by flow cytometry. G Proportions of DiBAC-positive HK-2 cells in the control and BaCl 2 (10 μM) groups with or without 25 μM polymyxin B treatment measured by flow cytometry ( n = 5 for WT and n = 4 for KOs). Data are shown as box and whisker plots. One-way (for WT) or two-way (for KOs) ANOVA was employed for multi-group comparisons. ** p

    Techniques Used: Flow Cytometry, Whisker Assay

    Knockout or inhibition of Kir4.2 and Kir5.1 prevented polymyxin-induced toxicity in HK-2 cells. A Western blot showing the expression levels of KCNJ15 and KCNJ16 after knockout; actin was used as an internal control. B Viability of wild-type HK-2, KCNJ15 KO and KCNJ16 KO cells following 24-h exposure to 10 and 25 µM polymyxin B ( n = 6). C Viability of HK-2 cells following 24-h exposure to 0–100 µM BaCl 2 with or without 25 µM polymyxin B ( n = 5). D Viability of HK-2 cells following the treatment of 0–25 µM VU0134992 alone or in combination with 25 µM polymyxin B for 24 h ( n = 3 for controls, and n = 4 for treatment groups). E Morphologies of wild-type, KNCJ15 KO, and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B or polymyxin B with the combination of 50 µM BaCl 2 , or 5 µM VU0134992 to wild-type cells . Two-way ANOVA was employed for multi-group comparisons and Tukey's multiple comparison test was employed for post-test. * p
    Figure Legend Snippet: Knockout or inhibition of Kir4.2 and Kir5.1 prevented polymyxin-induced toxicity in HK-2 cells. A Western blot showing the expression levels of KCNJ15 and KCNJ16 after knockout; actin was used as an internal control. B Viability of wild-type HK-2, KCNJ15 KO and KCNJ16 KO cells following 24-h exposure to 10 and 25 µM polymyxin B ( n = 6). C Viability of HK-2 cells following 24-h exposure to 0–100 µM BaCl 2 with or without 25 µM polymyxin B ( n = 5). D Viability of HK-2 cells following the treatment of 0–25 µM VU0134992 alone or in combination with 25 µM polymyxin B for 24 h ( n = 3 for controls, and n = 4 for treatment groups). E Morphologies of wild-type, KNCJ15 KO, and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B or polymyxin B with the combination of 50 µM BaCl 2 , or 5 µM VU0134992 to wild-type cells . Two-way ANOVA was employed for multi-group comparisons and Tukey's multiple comparison test was employed for post-test. * p

    Techniques Used: Knock-Out, Inhibition, Western Blot, Expressing

    2) Product Images from "Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa"

    Article Title: Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22115972

    Viability and progressive motility of spermatozoa was comparable in WT and KO mice. The box plots report the evaluation of viability ( A ) and progressive motility ( B ) for spermatozoa collected from WT and KO mice. Both viability and progressive motility were not significantly changed ( p > 0.05) by KO of Kir5.1 channels.
    Figure Legend Snippet: Viability and progressive motility of spermatozoa was comparable in WT and KO mice. The box plots report the evaluation of viability ( A ) and progressive motility ( B ) for spermatozoa collected from WT and KO mice. Both viability and progressive motility were not significantly changed ( p > 0.05) by KO of Kir5.1 channels.

    Techniques Used: Mouse Assay

    Expression of Kir4.1 and Kir5.1 channels in the cauda epididymis. ( A ) Kir4.1 is expressed in the epithelial cells lining the epididymal ducts (white arrow) and in peritubular smooth muscle of the cauda epididymis (red arrow). Immunoreactivity was absent in the lumen where spermatozoa are located, implying the lack of Kir4.1 expression in these cells. ( B ) Magnified image taken from the upper part of panel A. ( C ) Image showing strong expression of Kir5.1 in spermatozoa. ( D ) Magnification of the lumen of the cauda epididymis. Staining shows the localisation of Kir5.1 subunits in the head of the sperm. Scale bar in ( A ), ( B ), and ( C ) = 25 µm, and in ( D ) = 20 µm.
    Figure Legend Snippet: Expression of Kir4.1 and Kir5.1 channels in the cauda epididymis. ( A ) Kir4.1 is expressed in the epithelial cells lining the epididymal ducts (white arrow) and in peritubular smooth muscle of the cauda epididymis (red arrow). Immunoreactivity was absent in the lumen where spermatozoa are located, implying the lack of Kir4.1 expression in these cells. ( B ) Magnified image taken from the upper part of panel A. ( C ) Image showing strong expression of Kir5.1 in spermatozoa. ( D ) Magnification of the lumen of the cauda epididymis. Staining shows the localisation of Kir5.1 subunits in the head of the sperm. Scale bar in ( A ), ( B ), and ( C ) = 25 µm, and in ( D ) = 20 µm.

    Techniques Used: Expressing, Staining

    3) Product Images from "Glial and neuronal expression of the Inward Rectifying Potassium Channel Kir7.1 in the adult mouse brain"

    Article Title: Glial and neuronal expression of the Inward Rectifying Potassium Channel Kir7.1 in the adult mouse brain

    Journal: Journal of Anatomy

    doi: 10.1111/joa.13048

    Neurons and glia express Kir7.1 in vitro . (A, B) Cells were isolated from P1‐2 mouse cortex and analysed after 14 days in vitro (DIV) by double immunofluorescence labelling for Kir7.1 (green) and the astrocyte marker GFAP (red, A) or neuronal marker Tuj1 (red, B, asterisks indicate Kir7.1+Tuj1‐ cells that are most likely astrocytes (C‐F) Optic nerve glial explant cultures from P7‐12 mice were analysed at 10DIV by double immunofluorescence labelling for Kir7.1 (green), with the plasmalemmal markers Na + /K + ‐ATPase (C) and PSD95 (D), or the glial Kir channels Kir4.1 (E) and Kir5.1 (F), In all cases, overlays are illustrated (Ai, Bi, Ci, Di, Ei, Fi, co‐expression appears yellow), together with individual channels (Aii‐iii, Bii‐iii, Ci‐iii, Di‐iii, Ei‐iii, Fi‐iii). Scale Bars: A‐B = 50μm; C‐F = 20µm. [Colour figure can be viewed at wileyonlinelibrary.com ]
    Figure Legend Snippet: Neurons and glia express Kir7.1 in vitro . (A, B) Cells were isolated from P1‐2 mouse cortex and analysed after 14 days in vitro (DIV) by double immunofluorescence labelling for Kir7.1 (green) and the astrocyte marker GFAP (red, A) or neuronal marker Tuj1 (red, B, asterisks indicate Kir7.1+Tuj1‐ cells that are most likely astrocytes (C‐F) Optic nerve glial explant cultures from P7‐12 mice were analysed at 10DIV by double immunofluorescence labelling for Kir7.1 (green), with the plasmalemmal markers Na + /K + ‐ATPase (C) and PSD95 (D), or the glial Kir channels Kir4.1 (E) and Kir5.1 (F), In all cases, overlays are illustrated (Ai, Bi, Ci, Di, Ei, Fi, co‐expression appears yellow), together with individual channels (Aii‐iii, Bii‐iii, Ci‐iii, Di‐iii, Ei‐iii, Fi‐iii). Scale Bars: A‐B = 50μm; C‐F = 20µm. [Colour figure can be viewed at wileyonlinelibrary.com ]

    Techniques Used: In Vitro, Isolation, Immunofluorescence, Marker, Mouse Assay, Expressing

    4) Product Images from "Kir5.1 regulates Nedd4-2-mediated ubiquitination of Kir4.1 in distal nephron"

    Article Title: Kir5.1 regulates Nedd4-2-mediated ubiquitination of Kir4.1 in distal nephron

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00059.2018

    Kir 5.1 is required for Nedd4-2-mediated ubiquitination of Kir4.1. A : Ubiquitination assay shows ubiquitinated K channel in HEK293 cells transfected with Flag-tagged Kir4.1 or Kir5.1, Kir4.1+Nedd4-2, Kir4.1+Kir5.1, Kir5.1+Nedd4-2, and Kir4.1/5.1+ Nedd4-2, respectively. Flag antibody was used to immunoprecipitate Flag-tagged Kir4.1 or 5.1, and ubiquitin (Ub) antibody was used to detect ubiquitinated K channels (indicated by a bracket). B : Ubiquitination assay with SDS pretreatment shows ubiquitinated K channels in HEK293 cells transfected with Flag-tagged Kir4.1, Kir4.1+Nedd4-2, and Kir4.1/5.1+Nedd4-2. Lower panel shows the expression of Kir4.1 used for immunoprecipitation (IP). IB, immunoblot.
    Figure Legend Snippet: Kir 5.1 is required for Nedd4-2-mediated ubiquitination of Kir4.1. A : Ubiquitination assay shows ubiquitinated K channel in HEK293 cells transfected with Flag-tagged Kir4.1 or Kir5.1, Kir4.1+Nedd4-2, Kir4.1+Kir5.1, Kir5.1+Nedd4-2, and Kir4.1/5.1+ Nedd4-2, respectively. Flag antibody was used to immunoprecipitate Flag-tagged Kir4.1 or 5.1, and ubiquitin (Ub) antibody was used to detect ubiquitinated K channels (indicated by a bracket). B : Ubiquitination assay with SDS pretreatment shows ubiquitinated K channels in HEK293 cells transfected with Flag-tagged Kir4.1, Kir4.1+Nedd4-2, and Kir4.1/5.1+Nedd4-2. Lower panel shows the expression of Kir4.1 used for immunoprecipitation (IP). IB, immunoblot.

    Techniques Used: Ubiquitin Assay, Transfection, Expressing, Immunoprecipitation

    Deletion of Kir5.1 stimulates the basolateral Kir4.1 in the distal convoluted tubule (DCT). A : Whole-cell recording showing Ba 2+ -sensitive K currents in the DCT of the wild-type (WT) or Kir5.1 knockout (KO) mice. K currents were measured with a ramp protocol from −100 to 100 mV using symmetrical 145 mM K solution in the bath and pipette. B : Bar graph summarizes the results of experiments in which Ba 2+ -sensitive K currents of the DCT were measured at −60 mV from the WT and Kir5.1 KO mice ( n = 6). Western blot (IB) shows the expression of Kir4.1 in WT and Kir5.1 KO mice ( C ), and a bar graph summarizes normalized band density of Kir4.1 ( D ). E : Immunostaining of Kir4.1 in kidney from WT and Kir5.1 KO mice. Treatment for the kidney slides was identical. pA, picoamperes.
    Figure Legend Snippet: Deletion of Kir5.1 stimulates the basolateral Kir4.1 in the distal convoluted tubule (DCT). A : Whole-cell recording showing Ba 2+ -sensitive K currents in the DCT of the wild-type (WT) or Kir5.1 knockout (KO) mice. K currents were measured with a ramp protocol from −100 to 100 mV using symmetrical 145 mM K solution in the bath and pipette. B : Bar graph summarizes the results of experiments in which Ba 2+ -sensitive K currents of the DCT were measured at −60 mV from the WT and Kir5.1 KO mice ( n = 6). Western blot (IB) shows the expression of Kir4.1 in WT and Kir5.1 KO mice ( C ), and a bar graph summarizes normalized band density of Kir4.1 ( D ). E : Immunostaining of Kir4.1 in kidney from WT and Kir5.1 KO mice. Treatment for the kidney slides was identical. pA, picoamperes.

    Techniques Used: Knock-Out, Mouse Assay, Transferring, Western Blot, Expressing, Immunostaining

    Mutation of TPVT motif of Kir5.1 abolishes Nedd4-2-mediated inhibition of Kir4.1/5.1. A : Western blot (IB) shows the expression of Kir4.1 in HEK293 cells transfected with Ki4.1+Nedd4-2, Kir4.1/5.1+Nedd4-2, Kir4.1+Nedd4-1, and Kir4.1/5.1+Nedd4-1. The expression of Nedd4 was shown in the middle panel. B : Normalized band density of Kir4.1 expression is shown in a bar graph. C : Bar graph summarizes the results of experiments in which Ba 2+ -sensitive K currents were measured (at −60 mV) with whole-cell recording in HEK293 cells transfected with Kir4.1/5.1, Kir4.1/5.1+Nedd4-2, Kir4.1/5.1 T249A +Nedd4-2, and Kir4.1/5.1 T249D +Nedd4-2, respectively ( n = 6). D : Ba 2+ -sensitive K currents measured at −60 mV with whole-cell recording in HEK293 cells transfected with Kir4.1/5.1, Kir4.1/5.1 T249A , and Kir4.1/5.1 T249D ( n = 6). Patch-clamp experiments were performed 24 h after the transfection, and symmetrical 145 mM K was used for the pipette and the bath solution. pA, picoamperes; pF, picofarads.
    Figure Legend Snippet: Mutation of TPVT motif of Kir5.1 abolishes Nedd4-2-mediated inhibition of Kir4.1/5.1. A : Western blot (IB) shows the expression of Kir4.1 in HEK293 cells transfected with Ki4.1+Nedd4-2, Kir4.1/5.1+Nedd4-2, Kir4.1+Nedd4-1, and Kir4.1/5.1+Nedd4-1. The expression of Nedd4 was shown in the middle panel. B : Normalized band density of Kir4.1 expression is shown in a bar graph. C : Bar graph summarizes the results of experiments in which Ba 2+ -sensitive K currents were measured (at −60 mV) with whole-cell recording in HEK293 cells transfected with Kir4.1/5.1, Kir4.1/5.1+Nedd4-2, Kir4.1/5.1 T249A +Nedd4-2, and Kir4.1/5.1 T249D +Nedd4-2, respectively ( n = 6). D : Ba 2+ -sensitive K currents measured at −60 mV with whole-cell recording in HEK293 cells transfected with Kir4.1/5.1, Kir4.1/5.1 T249A , and Kir4.1/5.1 T249D ( n = 6). Patch-clamp experiments were performed 24 h after the transfection, and symmetrical 145 mM K was used for the pipette and the bath solution. pA, picoamperes; pF, picofarads.

    Techniques Used: Mutagenesis, Inhibition, Western Blot, Expressing, Transfection, Patch Clamp, Transferring

    A scheme illustrating the role of Kir5.1 as a binding partner in mediating Nedd4-2 E3 ligase dependent degradation of Kir4.1 in the distal convoluted tubule (DCT). A part of Kir5.1 sequence (from AA241 to 260) including TPVT motif is shown on the top. Ub, ubiquitin.
    Figure Legend Snippet: A scheme illustrating the role of Kir5.1 as a binding partner in mediating Nedd4-2 E3 ligase dependent degradation of Kir4.1 in the distal convoluted tubule (DCT). A part of Kir5.1 sequence (from AA241 to 260) including TPVT motif is shown on the top. Ub, ubiquitin.

    Techniques Used: Binding Assay, Sequencing

    Deletion of Nedd4-2 increases Kir4.1 expression. A : Bar graph summarizes the results of experiments in which Ba 2+ -sensitive K currents of the distal convoluted tubule (DCT) were measured at −60 mV from the wild-type (WT) and Ks-Nedd4-2 knockout (KO) mice ( n = 6). B : Western blots (IB) showed the expression of Kir4.1 and Kir5.1 in WT and Ks-Nedd4-2 KO mice. A bar graph summarizes normalized band density of Kir4.1 and Ki5.1 (right panel). C : immunostaining of Kir4.1 in kidney from Ks-Nedd4-2 KO mice and WT control (without doxycycline treated). pA, picoamperes.
    Figure Legend Snippet: Deletion of Nedd4-2 increases Kir4.1 expression. A : Bar graph summarizes the results of experiments in which Ba 2+ -sensitive K currents of the distal convoluted tubule (DCT) were measured at −60 mV from the wild-type (WT) and Ks-Nedd4-2 knockout (KO) mice ( n = 6). B : Western blots (IB) showed the expression of Kir4.1 and Kir5.1 in WT and Ks-Nedd4-2 KO mice. A bar graph summarizes normalized band density of Kir4.1 and Ki5.1 (right panel). C : immunostaining of Kir4.1 in kidney from Ks-Nedd4-2 KO mice and WT control (without doxycycline treated). pA, picoamperes.

    Techniques Used: Expressing, Knock-Out, Mouse Assay, Western Blot, Immunostaining

    Nedd4-2 interacts with Kir5.1. A : Coimmunoprecipitation experiments (CO-IP) show the interaction of Kir5.1 with Nedd4-2 or Nedd4-1 in HEK293 cells transfected with HA-GFP-Kir5.1 and Flag-tagged Nedd4-2 or Nedd4-1 ( top ). Equal amount of Flag-Nedd4-2 and -Nedd4-1 used for CO-IP ( middle ). Input of HA-GFP-Kir5.1 ( bottom ). B : CO-IP shows the interaction of Nedd4-2 or Nedd4-1 with Kir5.1 in HEK293 cells transfected with myc-Kir5.1 and Flag-tagged-Nedd4-2. An equal amount of myc-Kir5.1 used for CO-IP ( bottom ). *Unspecific band. Cells transfected with myc-Kir5.1 are used as negative control (labeled as Kir5.1). C : CO-IP shows that interaction of Nedd4-2 and Kir5.1/mutants in HEK293 cells transfected with Flag-tagged-Nedd4-2 and HA-GFP-Kir5.1 or mutants (Kir5.1 T249A or Kir5.1 T249D ) ( top ). Equal amount of Flag-Nedd4-2 and -Nedd4-1 used for CO-IP ( middle ). Input of HA-GFP-Kir5.1 ( bottom ). Cells transfected with empty vector are used as IgG control (labeled as IgG). Cells transfected with Flag-Nedd4-2 are used as negative control (labeled as Nedd4-2). D : CO-IP shows that interaction of Nedd4-2 and Kir5.1/mutants in HEK293 cells transfected with Flag-tagged-Nedd4-2 and HA-GFP-Kir5.1 or mutants (Kir5.1 T252A or Kir5.1 T252D ) ( top ). Amount of Flag-Nedd4-2 used for CO-IP ( middle ). Input of HA-GFP-Kir5.1 ( bottom ). E : bar graph illustrates the normalized band density for the results of CO-IP. GFP, green fluorescent protein; IB, immunoblot.
    Figure Legend Snippet: Nedd4-2 interacts with Kir5.1. A : Coimmunoprecipitation experiments (CO-IP) show the interaction of Kir5.1 with Nedd4-2 or Nedd4-1 in HEK293 cells transfected with HA-GFP-Kir5.1 and Flag-tagged Nedd4-2 or Nedd4-1 ( top ). Equal amount of Flag-Nedd4-2 and -Nedd4-1 used for CO-IP ( middle ). Input of HA-GFP-Kir5.1 ( bottom ). B : CO-IP shows the interaction of Nedd4-2 or Nedd4-1 with Kir5.1 in HEK293 cells transfected with myc-Kir5.1 and Flag-tagged-Nedd4-2. An equal amount of myc-Kir5.1 used for CO-IP ( bottom ). *Unspecific band. Cells transfected with myc-Kir5.1 are used as negative control (labeled as Kir5.1). C : CO-IP shows that interaction of Nedd4-2 and Kir5.1/mutants in HEK293 cells transfected with Flag-tagged-Nedd4-2 and HA-GFP-Kir5.1 or mutants (Kir5.1 T249A or Kir5.1 T249D ) ( top ). Equal amount of Flag-Nedd4-2 and -Nedd4-1 used for CO-IP ( middle ). Input of HA-GFP-Kir5.1 ( bottom ). Cells transfected with empty vector are used as IgG control (labeled as IgG). Cells transfected with Flag-Nedd4-2 are used as negative control (labeled as Nedd4-2). D : CO-IP shows that interaction of Nedd4-2 and Kir5.1/mutants in HEK293 cells transfected with Flag-tagged-Nedd4-2 and HA-GFP-Kir5.1 or mutants (Kir5.1 T252A or Kir5.1 T252D ) ( top ). Amount of Flag-Nedd4-2 used for CO-IP ( middle ). Input of HA-GFP-Kir5.1 ( bottom ). E : bar graph illustrates the normalized band density for the results of CO-IP. GFP, green fluorescent protein; IB, immunoblot.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Negative Control, Labeling, Plasmid Preparation

    Nedd4-2 but not Nedd4-1 inhibits Kir4.1/Kir5.1 channels. Whole-cell recording shows Ba 2+ -sensitive K currents measured from −60 mV to 60 mV at 20-mV steps in HEK293 cells transfected with Kir4.1+Kir5.1 ( A ) or with Kir4.1/5.1 +Nedd4-2 ( B ). The symmetrical 145 mM K solution was used for both bath and pipette. C : Bar graph summarizes the results of experiments ( n = 6) in which Ba 2+ -sensitive K currents were measured with whole-cell recording in HEK293 cells transfected with Kir4.1, Kir4.1+Nedd4-2, Kir4.1/5.1, Kir4.1/5.1+Nedd4-2, Kir4.1/5.1+Nedd4-1, and Kir4.1/5.1+dead Nedd4-2. The patch-clamp experiments were performed 24 h after the transfection and the positive transfected cells were identified by green fluorescent protein (GFP) fluorescence. pA, picoamperes; pF, picofarads.
    Figure Legend Snippet: Nedd4-2 but not Nedd4-1 inhibits Kir4.1/Kir5.1 channels. Whole-cell recording shows Ba 2+ -sensitive K currents measured from −60 mV to 60 mV at 20-mV steps in HEK293 cells transfected with Kir4.1+Kir5.1 ( A ) or with Kir4.1/5.1 +Nedd4-2 ( B ). The symmetrical 145 mM K solution was used for both bath and pipette. C : Bar graph summarizes the results of experiments ( n = 6) in which Ba 2+ -sensitive K currents were measured with whole-cell recording in HEK293 cells transfected with Kir4.1, Kir4.1+Nedd4-2, Kir4.1/5.1, Kir4.1/5.1+Nedd4-2, Kir4.1/5.1+Nedd4-1, and Kir4.1/5.1+dead Nedd4-2. The patch-clamp experiments were performed 24 h after the transfection and the positive transfected cells were identified by green fluorescent protein (GFP) fluorescence. pA, picoamperes; pF, picofarads.

    Techniques Used: Transfection, Transferring, Patch Clamp, Fluorescence

    5) Product Images from "Oligodendrocyte-encoded Kir4.1 function is required for axonal integrity"

    Article Title: Oligodendrocyte-encoded Kir4.1 function is required for axonal integrity

    Journal: eLife

    doi: 10.7554/eLife.36428

    Validation of OL-encoded Kcnj10 cKO efficiency Kir4.1 channels were efficiently ablated from ON OLs in cKO-1 (n = 5) and cKO-2 (n = 4) mice versus control ONs (n = 5; A ). One-way ANOVA with Tukey’s multiple comparisons test was performed in A ; ****p≤0.0001. Kcnj10 was upregulated during OL differentiation, and expression significantly suppressed in purified and immunopanned OPCs (ctrl: n = 3, cKO-1: n = 3) and OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice ( B ). Conversely, Kcnj16 was not downregulated in OPCs (ctrl: n = 3, cKO-1: n = 3) and OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice in vitro, however, note Kcnj16 downregulation during OPC-OL maturation ( C ). Cacna1c mRNA levels were increased in cultured OPCs (ctrl: n = 3, cKO-1: n = 3) but not OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice suggesting a partial activation of Cav1.2 channels in OPCs ( D ). One-way ANOVA with Tukey’s multiple comparison tests were performed in B–D ; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. No difference in outer tongue Kir4.1 IEM labeling between controls and ON cKO-1 tissue ( E ). Trend towards decreased Kir4.1 IEM labeling in cKO-2 ON tissue as compared to controls with respect to myelin compartments but not AS fibers ( F ). Background IEM labeling without primary antibody confirmed specificity of Kir4.1 IEM antibody labeling of myelin compartments and astrocyte fibers ( G ). Mann-Whitney tests were performed in E–G ; ****p≤0.0001, p=0.8 ( E , outer tongue IEM), p=0.07 ( F , inner tongue IEM), p=0.6 ( F , compact myelin IEM), p=0.3 ( F , outer tongue IEM), p=0.81 ( F , AS fiber IEM), p=0.12 ( G , compact myelin IEM), p=0.08 ( G , outer tongue IEM), p=0.03 ( G , AS fiber IEM). Data are presented as mean ±s.e.m in A–G .
    Figure Legend Snippet: Validation of OL-encoded Kcnj10 cKO efficiency Kir4.1 channels were efficiently ablated from ON OLs in cKO-1 (n = 5) and cKO-2 (n = 4) mice versus control ONs (n = 5; A ). One-way ANOVA with Tukey’s multiple comparisons test was performed in A ; ****p≤0.0001. Kcnj10 was upregulated during OL differentiation, and expression significantly suppressed in purified and immunopanned OPCs (ctrl: n = 3, cKO-1: n = 3) and OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice ( B ). Conversely, Kcnj16 was not downregulated in OPCs (ctrl: n = 3, cKO-1: n = 3) and OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice in vitro, however, note Kcnj16 downregulation during OPC-OL maturation ( C ). Cacna1c mRNA levels were increased in cultured OPCs (ctrl: n = 3, cKO-1: n = 3) but not OLs (ctrl: n = 3, cKO-1: n = 3) from cKO-1 mice suggesting a partial activation of Cav1.2 channels in OPCs ( D ). One-way ANOVA with Tukey’s multiple comparison tests were performed in B–D ; *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001. No difference in outer tongue Kir4.1 IEM labeling between controls and ON cKO-1 tissue ( E ). Trend towards decreased Kir4.1 IEM labeling in cKO-2 ON tissue as compared to controls with respect to myelin compartments but not AS fibers ( F ). Background IEM labeling without primary antibody confirmed specificity of Kir4.1 IEM antibody labeling of myelin compartments and astrocyte fibers ( G ). Mann-Whitney tests were performed in E–G ; ****p≤0.0001, p=0.8 ( E , outer tongue IEM), p=0.07 ( F , inner tongue IEM), p=0.6 ( F , compact myelin IEM), p=0.3 ( F , outer tongue IEM), p=0.81 ( F , AS fiber IEM), p=0.12 ( G , compact myelin IEM), p=0.08 ( G , outer tongue IEM), p=0.03 ( G , AS fiber IEM). Data are presented as mean ±s.e.m in A–G .

    Techniques Used: Mouse Assay, Expressing, Purification, In Vitro, Cell Culture, Activation Assay, Labeling, Antibody Labeling, MANN-WHITNEY

    6) Product Images from "Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels"

    Article Title: Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00034

    Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P
    Figure Legend Snippet: Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P

    Techniques Used: Expressing

    Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).
    Figure Legend Snippet: Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Isolation, Quantitative RT-PCR

    Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.
    Figure Legend Snippet: Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.

    Techniques Used: Expressing, Immunofluorescence, Labeling

    Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .
    Figure Legend Snippet: Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .

    Techniques Used: Labeling

    7) Product Images from "Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels"

    Article Title: Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00034

    Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P
    Figure Legend Snippet: Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P

    Techniques Used: Expressing

    Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).
    Figure Legend Snippet: Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Isolation, Quantitative RT-PCR

    Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.
    Figure Legend Snippet: Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.

    Techniques Used: Expressing, Immunofluorescence, Labeling

    Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .
    Figure Legend Snippet: Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .

    Techniques Used: Labeling

    8) Product Images from "Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels"

    Article Title: Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00034

    Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P
    Figure Legend Snippet: Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P

    Techniques Used: Expressing

    Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).
    Figure Legend Snippet: Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Isolation, Quantitative RT-PCR

    Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.
    Figure Legend Snippet: Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.

    Techniques Used: Expressing, Immunofluorescence, Labeling

    Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .
    Figure Legend Snippet: Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .

    Techniques Used: Labeling

    9) Product Images from "Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels"

    Article Title: Identifying Candidate Genes that Underlie Cellular pH Sensitivity in Serotonin Neurons Using Transcriptomics: A Potential Role for Kir5.1 Channels

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00034

    Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P
    Figure Legend Snippet: Age-related changes in the expression of select K + channels the rostral medullary raphe. (A) Kcnj10, Kcnj16, Kcnk3, Kcnk9 , and Kcna2 gene expression patterns across age (P0, 7, 19, and adult) within the rostral raphe ( ∗ P

    Techniques Used: Expressing

    Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).
    Figure Legend Snippet: Single cell qPCR from individual 5-HT neurons confirmed Kcnj10 and Kcnj16 gene expression. (A) Representative visualization of eGFP-expressing brainstem 5-HT neuron and the isolation of the intracellular contents. (B) Number of single 5-HT neuron samples that did not express Kcnj10 Kcnj16 (red), that only expressed Kcnj10 (pink), only Kcnj16 (blue), and both Kcnj10 and Kcnj16 (green). (C) Representative expression pattern of 5-HT specific ( Tph2 and Ddc ), neuronal specific ( Map2 and Nefl ), and glial specific ( Gfap, Aqp4 , and Sox10 ) gene markers from a bulk raphe tissue homogenate normalized to Tph2 expression. (D) Expression pattern of 5-HT specific, neuronal specific, and glial specific genes among single 5-HT neurons normalized to Tph2 demonstrating purity and specificity of single cell intracellular content isolation using single cell qRT-PCR ( n = 7; ∗ vs. Gfap , Aqp4 , and Sox10 by One-Way ANOVA). (E) Single cell qRT-PCR C t-values demonstrating 5-HT specific expression of K + channel genes, including Kcnj10 and Kcnj16 ( n = 7).

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Isolation, Quantitative RT-PCR

    Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.
    Figure Legend Snippet: Kir4.1 and Kir5.1 expression in the medullary raphe. Immunofluorescence of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (A–F) or Kir5.1 (G–L) along with neuronal (NeuN) and astrocytic (GFAP) markers. Kir4.1 is expressed in medullary raphe astrocytes (A–C) , but not neurons (D–F) . Kir5.1 is expressed in medullary raphe astrocytes (G–I) and neurons (J–L) . The correlation coefficients for co-localization were as follows: C = 0.926, F = -0.063, I = 0.888, and L = 0.608. Images were obtained at 20×, and scale bars = 20 μm.

    Techniques Used: Expressing, Immunofluorescence, Labeling

    Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .
    Figure Legend Snippet: Kir5.1 but not Kir4.1 protein is co-localized to brainstem 5-HT neurons. Epifluorescence images of fixed-frozen brainstem sections labeled with primary antibodies targeting Kir4.1 (green; A ) and TPH (red; B ) or Kir5.1 (green; D ) and TPH (red; E ). Overlay images of indicate that Kir5.1 (correlation coefficient = 0.605; F ) but not Kir4.1 (correlation coefficient = 0.109; C ) is co-localized to TPH + 5-HT neurons (yellow), where TPH and Kir5.1 co-localization appears primarily perinuclear and to a lesser extent along the plasma membrane (G–I) . Confocal images (63×, 2.5 magnification) of brainstem tissues double-labeled with TPH (red; G ) and Kir5.1 (green; H ) also show intense co-localization (yellow; I ) along the perinuclear and to a lesser extent plasma membrane regions. Scale bar = 20 μm (C,F) or 10 μm (I) .

    Techniques Used: Labeling

    10) Product Images from "Expression of Kir4.1 and Kir5.1 inwardly rectifying potassium channels in oligodendrocytes, the myelinating cells of the CNS"

    Article Title: Expression of Kir4.1 and Kir5.1 inwardly rectifying potassium channels in oligodendrocytes, the myelinating cells of the CNS

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-016-1199-8

    Glial Kir5.1 expression is reduced in the absence of Kir4.1 subunit. Immunolabelling for Kir5.1 was determined in optic nerve explants cultures, comparing wild-type mice ( A , Kir4.1 +/+ ) with Kir4.1 knock-out mice ( B , Kir4.1 −/− ), and following transfection with scrambled shRNA ( C ) or Kir4.1 shRNA ( D ); transfected cells were identified by the expression of GFP (appears green ) and insets demonstrate Kir4.1 expression in controls ( Ai , Ci ) and complete ablation in Kir4.1 −/− mice ( Bi ) and Kir4.1 shRNA ( Di ). Scale bars 10 μm. Quantification of expression of Kir4.1 ( E ) and Kir5.1 ( F ) in Kir4.1 +/+ , Kir4.1 −/− , scrambled control and Kir4.1shRNA glia; analysis was performed on 10–12 cells in each group, and data are expressed as mean ± SEM number of voxels per µm 3 , *** p
    Figure Legend Snippet: Glial Kir5.1 expression is reduced in the absence of Kir4.1 subunit. Immunolabelling for Kir5.1 was determined in optic nerve explants cultures, comparing wild-type mice ( A , Kir4.1 +/+ ) with Kir4.1 knock-out mice ( B , Kir4.1 −/− ), and following transfection with scrambled shRNA ( C ) or Kir4.1 shRNA ( D ); transfected cells were identified by the expression of GFP (appears green ) and insets demonstrate Kir4.1 expression in controls ( Ai , Ci ) and complete ablation in Kir4.1 −/− mice ( Bi ) and Kir4.1 shRNA ( Di ). Scale bars 10 μm. Quantification of expression of Kir4.1 ( E ) and Kir5.1 ( F ) in Kir4.1 +/+ , Kir4.1 −/− , scrambled control and Kir4.1shRNA glia; analysis was performed on 10–12 cells in each group, and data are expressed as mean ± SEM number of voxels per µm 3 , *** p

    Techniques Used: Expressing, Mouse Assay, Knock-Out, Transfection, shRNA

    Functional implications of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels in oligodendrocytes. Oligodendroglial expression of Kir4.1 channels indicates they may be important in uptake of excess K + released during axonal action potential propagation, a function largely attribiuted to astrocytes. Due to their wrapping of axons, oligodendrocytes are exposed to large ionic and pH shifts during axonal electrical activity, and it is likely weakly rectifying homomeric Kir4.1 and strongly rectifying Kir4.1/Kir5.1 heteromeric channels are important in maintaining the negative resting membrane potential, which is essential for oligodendroglial and myelin integrity. Weakly rectifying homomeric Kir4.1 channels may preferentially extrude K + and supply extracellular K + for the Na + –K + -pumps, as described in transporting epithelia. In contrast, the pH sensitivity of heteromeric Kir4.1/Kir5.1 channels is likely to have a role in the CO 2 /pH chemosensation in glia, involving carbonic anhydrase that is enriched in astrocytes and oligodendrocytes. Furthermore, intracellular acidification and inhibition of Kir4.1/Kir5.1 channels has been shown to trigger release of ATP from astrocytes, which would act on oligodendroglial P2X and P2Y receptors to provide a mechanism of astrocyte–oligodendrocyte signaling in response to metabolic challenges, which has important implications for white matter physiology and pathology
    Figure Legend Snippet: Functional implications of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels in oligodendrocytes. Oligodendroglial expression of Kir4.1 channels indicates they may be important in uptake of excess K + released during axonal action potential propagation, a function largely attribiuted to astrocytes. Due to their wrapping of axons, oligodendrocytes are exposed to large ionic and pH shifts during axonal electrical activity, and it is likely weakly rectifying homomeric Kir4.1 and strongly rectifying Kir4.1/Kir5.1 heteromeric channels are important in maintaining the negative resting membrane potential, which is essential for oligodendroglial and myelin integrity. Weakly rectifying homomeric Kir4.1 channels may preferentially extrude K + and supply extracellular K + for the Na + –K + -pumps, as described in transporting epithelia. In contrast, the pH sensitivity of heteromeric Kir4.1/Kir5.1 channels is likely to have a role in the CO 2 /pH chemosensation in glia, involving carbonic anhydrase that is enriched in astrocytes and oligodendrocytes. Furthermore, intracellular acidification and inhibition of Kir4.1/Kir5.1 channels has been shown to trigger release of ATP from astrocytes, which would act on oligodendroglial P2X and P2Y receptors to provide a mechanism of astrocyte–oligodendrocyte signaling in response to metabolic challenges, which has important implications for white matter physiology and pathology

    Techniques Used: Functional Assay, Expressing, Activity Assay, Inhibition

    Specific reduction in plasmalemmal Kir5.1 in the absence of Kir4.1. Immunolocalization of Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explant astrocytes identified by expression of GFAP, following transfection with scrambled shRNA ( A ) or Kir4.1 shRNA ( B ); transfected cells were identified by co-transfection with GFP (appears green ) and the co-localization channel indicates voxels in which Kir5.1 and Na–K-ATPase immunolabelling was at the same intensity ( Avi , Bvi ). Scale bars 20 μm. C Quantification of plasmalemmal Kir5.1 expressed as percentage of total Kir5.1 + voxels (data are mean ± SEM, n = 11–13 per group; * p
    Figure Legend Snippet: Specific reduction in plasmalemmal Kir5.1 in the absence of Kir4.1. Immunolocalization of Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explant astrocytes identified by expression of GFAP, following transfection with scrambled shRNA ( A ) or Kir4.1 shRNA ( B ); transfected cells were identified by co-transfection with GFP (appears green ) and the co-localization channel indicates voxels in which Kir5.1 and Na–K-ATPase immunolabelling was at the same intensity ( Avi , Bvi ). Scale bars 20 μm. C Quantification of plasmalemmal Kir5.1 expressed as percentage of total Kir5.1 + voxels (data are mean ± SEM, n = 11–13 per group; * p

    Techniques Used: Expressing, Transfection, shRNA, Cotransfection

    Reduction of Kir5.1 in oligodendrocytes and myelin in the absence of Kir4.1. Immunolocalization of Kir5.1 with myelin basic protein, MBP ( A , B ) and the oligodenrocyte marker APC/CC1 ( C – F ), in brain tissue from wild-type Kir4.1 +/+ mice ( A , C , E ) compared to Kir4.1 −/− knock-out mice ( B , D , F ). Scale bars 20 μm. Western blot analysis of Kir5.1 from total lysates of optic nerve ( G ) and brain ( H ) from wild-type Kir4.1 +/+ and Kir4.1 −/− knock-out mice, and mean (±SEM) integrated density normalised against β-actin ( I , n = 3, ** p
    Figure Legend Snippet: Reduction of Kir5.1 in oligodendrocytes and myelin in the absence of Kir4.1. Immunolocalization of Kir5.1 with myelin basic protein, MBP ( A , B ) and the oligodenrocyte marker APC/CC1 ( C – F ), in brain tissue from wild-type Kir4.1 +/+ mice ( A , C , E ) compared to Kir4.1 −/− knock-out mice ( B , D , F ). Scale bars 20 μm. Western blot analysis of Kir5.1 from total lysates of optic nerve ( G ) and brain ( H ) from wild-type Kir4.1 +/+ and Kir4.1 −/− knock-out mice, and mean (±SEM) integrated density normalised against β-actin ( I , n = 3, ** p

    Techniques Used: Marker, Mouse Assay, Knock-Out, Western Blot

    Expression of Kir4.1 and Kir5.1 in oligodendrocytes and astrocytes in the cerebellum. Immunolabelling for Kir4.1 and Kir5.1, in combination with GFAP for astrocytes ( A , C ), and APC/CC1 for oligodendrocytes ( B , D ). Immunolabelling for Kir4.1 ( E ) and Kir5.1 ( F ) in mice in which EGFP is under the control of the oligodendrocyte-specific Sox10 promoter. G Double immunolabelling for Kir4.1 ( red ) and the oligodenrocyte-specific marker Olig2 ( green ). Insets in Aiv and Civ illustrate negative controls, in the Kir4.1 KO mouse ( Aiv ) and following preincubation with the Kir5.1 blocking peptide ( Civ ). Scale bars 20 μm. Western blot analysis of the brain and optic and nerve for Kir4.1 ( I ) and Kir5.1 ( J ); bands were absent in the negative controls, in the Kir4.1 knock-out mouse ( I ) following preincubation in the Kir5.1 blocking peptide ( J )
    Figure Legend Snippet: Expression of Kir4.1 and Kir5.1 in oligodendrocytes and astrocytes in the cerebellum. Immunolabelling for Kir4.1 and Kir5.1, in combination with GFAP for astrocytes ( A , C ), and APC/CC1 for oligodendrocytes ( B , D ). Immunolabelling for Kir4.1 ( E ) and Kir5.1 ( F ) in mice in which EGFP is under the control of the oligodendrocyte-specific Sox10 promoter. G Double immunolabelling for Kir4.1 ( red ) and the oligodenrocyte-specific marker Olig2 ( green ). Insets in Aiv and Civ illustrate negative controls, in the Kir4.1 KO mouse ( Aiv ) and following preincubation with the Kir5.1 blocking peptide ( Civ ). Scale bars 20 μm. Western blot analysis of the brain and optic and nerve for Kir4.1 ( I ) and Kir5.1 ( J ); bands were absent in the negative controls, in the Kir4.1 knock-out mouse ( I ) following preincubation in the Kir5.1 blocking peptide ( J )

    Techniques Used: Expressing, Mouse Assay, Marker, Blocking Assay, Western Blot, Knock-Out

    Expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Immunolabelling for Kir4.1 ( A , C ) and Kir5.1 ( B , D ), in GFAP-GFP mice to identify astrocytes ( A , B ) and PLP-DsRED mice to identify oligodendrocytes ( C , D ). Cellular expression of Kir4.1 and Kir5.1 is demonstrated by the generation of colocalisation channels ( Av , Bv , Cv , Dv ) from confocal z -stacks ( Aiv , Biv , Civ , Div ), and green and red channels of equal intensity appear yellow . Scale bars 20 μm
    Figure Legend Snippet: Expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Immunolabelling for Kir4.1 ( A , C ) and Kir5.1 ( B , D ), in GFAP-GFP mice to identify astrocytes ( A , B ) and PLP-DsRED mice to identify oligodendrocytes ( C , D ). Cellular expression of Kir4.1 and Kir5.1 is demonstrated by the generation of colocalisation channels ( Av , Bv , Cv , Dv ) from confocal z -stacks ( Aiv , Biv , Civ , Div ), and green and red channels of equal intensity appear yellow . Scale bars 20 μm

    Techniques Used: Expressing, Mouse Assay, Plasmid Purification

    Co-expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Co-immunolocalization of Kir4.1 and Kir5.1 in optic nerve explant cultures, in astrocytes identified by GFAP immunolabelling ( A ) and oligodendrocytes identified by PLP-DsRED ( B ). The overlay and individual channels are illustrated, together with the co-localisation channel for Kir4.1/Kir5.1 ( Aii, Bii ). Boxed areas on overlay images ( Ai , Bi ) are enlarged in Avi – Aviii and Bvi – Bviii , to illustrate punctate colocalization of Kir4.1 and Kir5.1 along processes (some indicated by arrows ). Scale bars 20 μm. Quantification of the number of voxels that were positive for Kir4.1 and Kir5.1 alone and of Kir4.1/Kir5.1 together, in astrocytes ( C , n = 15) and oligodendrocytes ( D , n = 13); data are mean ± SEM. Co-immunoprecipitation of Kir4.1 with Kir5.1 ( E ) and of Kir5.1 with Kir4.1 ( F ) from total brain and optic nerve (ON) lysates; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1, and using the blocking peptide for Kir5.1
    Figure Legend Snippet: Co-expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Co-immunolocalization of Kir4.1 and Kir5.1 in optic nerve explant cultures, in astrocytes identified by GFAP immunolabelling ( A ) and oligodendrocytes identified by PLP-DsRED ( B ). The overlay and individual channels are illustrated, together with the co-localisation channel for Kir4.1/Kir5.1 ( Aii, Bii ). Boxed areas on overlay images ( Ai , Bi ) are enlarged in Avi – Aviii and Bvi – Bviii , to illustrate punctate colocalization of Kir4.1 and Kir5.1 along processes (some indicated by arrows ). Scale bars 20 μm. Quantification of the number of voxels that were positive for Kir4.1 and Kir5.1 alone and of Kir4.1/Kir5.1 together, in astrocytes ( C , n = 15) and oligodendrocytes ( D , n = 13); data are mean ± SEM. Co-immunoprecipitation of Kir4.1 with Kir5.1 ( E ) and of Kir5.1 with Kir4.1 ( F ) from total brain and optic nerve (ON) lysates; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1, and using the blocking peptide for Kir5.1

    Techniques Used: Expressing, Plasmid Purification, Immunoprecipitation, Knock-Out, Mouse Assay, Blocking Assay

    Plasmalemmal expression of Kir4.1 and Kir5.1 subunit in optic nerve glia. Immunolocalization of Kir4.1 and Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explants of astrocytes identified by GFAP ( A , B ) and oligodendrocytes identified by PLP-DsRed ( C , D ). Scale bars 20 μm. Quantification in astrocytes and oligodendrocytes of total number of voxels immunopositive for Kir4.1 and Kir5.1, compared to voxels that were identified as colocalized for Kir4.1/Na–K-ATPase ( E ) and Kir5.1/Na–K-ATPase ( F ); data are mean ± SEM, n = 13 cells for each analysis. Western blot analysis of Kir5.1 ( G ) and Kir4.1 ( H ) in total optic nerve lysate and plasma membrane fraction. Co-immunoprecipitation of Kir4.1 ( I ) and Kir5.1 ( J ) with PSD95, in total brain and optic nerve (ON) lysate; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1 and preincubation with the blocking peptide for Kir5.1
    Figure Legend Snippet: Plasmalemmal expression of Kir4.1 and Kir5.1 subunit in optic nerve glia. Immunolocalization of Kir4.1 and Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explants of astrocytes identified by GFAP ( A , B ) and oligodendrocytes identified by PLP-DsRed ( C , D ). Scale bars 20 μm. Quantification in astrocytes and oligodendrocytes of total number of voxels immunopositive for Kir4.1 and Kir5.1, compared to voxels that were identified as colocalized for Kir4.1/Na–K-ATPase ( E ) and Kir5.1/Na–K-ATPase ( F ); data are mean ± SEM, n = 13 cells for each analysis. Western blot analysis of Kir5.1 ( G ) and Kir4.1 ( H ) in total optic nerve lysate and plasma membrane fraction. Co-immunoprecipitation of Kir4.1 ( I ) and Kir5.1 ( J ) with PSD95, in total brain and optic nerve (ON) lysate; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1 and preincubation with the blocking peptide for Kir5.1

    Techniques Used: Expressing, Plasmid Purification, Western Blot, Immunoprecipitation, Knock-Out, Mouse Assay, Blocking Assay

    11) Product Images from "Expression of Kir4.1 and Kir5.1 inwardly rectifying potassium channels in oligodendrocytes, the myelinating cells of the CNS"

    Article Title: Expression of Kir4.1 and Kir5.1 inwardly rectifying potassium channels in oligodendrocytes, the myelinating cells of the CNS

    Journal: Brain Structure & Function

    doi: 10.1007/s00429-016-1199-8

    Glial Kir5.1 expression is reduced in the absence of Kir4.1 subunit. Immunolabelling for Kir5.1 was determined in optic nerve explants cultures, comparing wild-type mice ( A , Kir4.1 +/+ ) with Kir4.1 knock-out mice ( B , Kir4.1 −/− ), and following transfection with scrambled shRNA ( C ) or Kir4.1 shRNA ( D ); transfected cells were identified by the expression of GFP (appears green ) and insets demonstrate Kir4.1 expression in controls ( Ai , Ci ) and complete ablation in Kir4.1 −/− mice ( Bi ) and Kir4.1 shRNA ( Di ). Scale bars 10 μm. Quantification of expression of Kir4.1 ( E ) and Kir5.1 ( F ) in Kir4.1 +/+ , Kir4.1 −/− , scrambled control and Kir4.1shRNA glia; analysis was performed on 10–12 cells in each group, and data are expressed as mean ± SEM number of voxels per µm 3 , *** p
    Figure Legend Snippet: Glial Kir5.1 expression is reduced in the absence of Kir4.1 subunit. Immunolabelling for Kir5.1 was determined in optic nerve explants cultures, comparing wild-type mice ( A , Kir4.1 +/+ ) with Kir4.1 knock-out mice ( B , Kir4.1 −/− ), and following transfection with scrambled shRNA ( C ) or Kir4.1 shRNA ( D ); transfected cells were identified by the expression of GFP (appears green ) and insets demonstrate Kir4.1 expression in controls ( Ai , Ci ) and complete ablation in Kir4.1 −/− mice ( Bi ) and Kir4.1 shRNA ( Di ). Scale bars 10 μm. Quantification of expression of Kir4.1 ( E ) and Kir5.1 ( F ) in Kir4.1 +/+ , Kir4.1 −/− , scrambled control and Kir4.1shRNA glia; analysis was performed on 10–12 cells in each group, and data are expressed as mean ± SEM number of voxels per µm 3 , *** p

    Techniques Used: Expressing, Mouse Assay, Knock-Out, Transfection, shRNA

    Functional implications of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels in oligodendrocytes. Oligodendroglial expression of Kir4.1 channels indicates they may be important in uptake of excess K + released during axonal action potential propagation, a function largely attribiuted to astrocytes. Due to their wrapping of axons, oligodendrocytes are exposed to large ionic and pH shifts during axonal electrical activity, and it is likely weakly rectifying homomeric Kir4.1 and strongly rectifying Kir4.1/Kir5.1 heteromeric channels are important in maintaining the negative resting membrane potential, which is essential for oligodendroglial and myelin integrity. Weakly rectifying homomeric Kir4.1 channels may preferentially extrude K + and supply extracellular K + for the Na + –K + -pumps, as described in transporting epithelia. In contrast, the pH sensitivity of heteromeric Kir4.1/Kir5.1 channels is likely to have a role in the CO 2 /pH chemosensation in glia, involving carbonic anhydrase that is enriched in astrocytes and oligodendrocytes. Furthermore, intracellular acidification and inhibition of Kir4.1/Kir5.1 channels has been shown to trigger release of ATP from astrocytes, which would act on oligodendroglial P2X and P2Y receptors to provide a mechanism of astrocyte–oligodendrocyte signaling in response to metabolic challenges, which has important implications for white matter physiology and pathology
    Figure Legend Snippet: Functional implications of homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels in oligodendrocytes. Oligodendroglial expression of Kir4.1 channels indicates they may be important in uptake of excess K + released during axonal action potential propagation, a function largely attribiuted to astrocytes. Due to their wrapping of axons, oligodendrocytes are exposed to large ionic and pH shifts during axonal electrical activity, and it is likely weakly rectifying homomeric Kir4.1 and strongly rectifying Kir4.1/Kir5.1 heteromeric channels are important in maintaining the negative resting membrane potential, which is essential for oligodendroglial and myelin integrity. Weakly rectifying homomeric Kir4.1 channels may preferentially extrude K + and supply extracellular K + for the Na + –K + -pumps, as described in transporting epithelia. In contrast, the pH sensitivity of heteromeric Kir4.1/Kir5.1 channels is likely to have a role in the CO 2 /pH chemosensation in glia, involving carbonic anhydrase that is enriched in astrocytes and oligodendrocytes. Furthermore, intracellular acidification and inhibition of Kir4.1/Kir5.1 channels has been shown to trigger release of ATP from astrocytes, which would act on oligodendroglial P2X and P2Y receptors to provide a mechanism of astrocyte–oligodendrocyte signaling in response to metabolic challenges, which has important implications for white matter physiology and pathology

    Techniques Used: Functional Assay, Expressing, Activity Assay, Inhibition

    Specific reduction in plasmalemmal Kir5.1 in the absence of Kir4.1. Immunolocalization of Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explant astrocytes identified by expression of GFAP, following transfection with scrambled shRNA ( A ) or Kir4.1 shRNA ( B ); transfected cells were identified by co-transfection with GFP (appears green ) and the co-localization channel indicates voxels in which Kir5.1 and Na–K-ATPase immunolabelling was at the same intensity ( Avi , Bvi ). Scale bars 20 μm. C Quantification of plasmalemmal Kir5.1 expressed as percentage of total Kir5.1 + voxels (data are mean ± SEM, n = 11–13 per group; * p
    Figure Legend Snippet: Specific reduction in plasmalemmal Kir5.1 in the absence of Kir4.1. Immunolocalization of Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explant astrocytes identified by expression of GFAP, following transfection with scrambled shRNA ( A ) or Kir4.1 shRNA ( B ); transfected cells were identified by co-transfection with GFP (appears green ) and the co-localization channel indicates voxels in which Kir5.1 and Na–K-ATPase immunolabelling was at the same intensity ( Avi , Bvi ). Scale bars 20 μm. C Quantification of plasmalemmal Kir5.1 expressed as percentage of total Kir5.1 + voxels (data are mean ± SEM, n = 11–13 per group; * p

    Techniques Used: Expressing, Transfection, shRNA, Cotransfection

    Reduction of Kir5.1 in oligodendrocytes and myelin in the absence of Kir4.1. Immunolocalization of Kir5.1 with myelin basic protein, MBP ( A , B ) and the oligodenrocyte marker APC/CC1 ( C – F ), in brain tissue from wild-type Kir4.1 +/+ mice ( A , C , E ) compared to Kir4.1 −/− knock-out mice ( B , D , F ). Scale bars 20 μm. Western blot analysis of Kir5.1 from total lysates of optic nerve ( G ) and brain ( H ) from wild-type Kir4.1 +/+ and Kir4.1 −/− knock-out mice, and mean (±SEM) integrated density normalised against β-actin ( I , n = 3, ** p
    Figure Legend Snippet: Reduction of Kir5.1 in oligodendrocytes and myelin in the absence of Kir4.1. Immunolocalization of Kir5.1 with myelin basic protein, MBP ( A , B ) and the oligodenrocyte marker APC/CC1 ( C – F ), in brain tissue from wild-type Kir4.1 +/+ mice ( A , C , E ) compared to Kir4.1 −/− knock-out mice ( B , D , F ). Scale bars 20 μm. Western blot analysis of Kir5.1 from total lysates of optic nerve ( G ) and brain ( H ) from wild-type Kir4.1 +/+ and Kir4.1 −/− knock-out mice, and mean (±SEM) integrated density normalised against β-actin ( I , n = 3, ** p

    Techniques Used: Marker, Mouse Assay, Knock-Out, Western Blot

    Expression of Kir4.1 and Kir5.1 in oligodendrocytes and astrocytes in the cerebellum. Immunolabelling for Kir4.1 and Kir5.1, in combination with GFAP for astrocytes ( A , C ), and APC/CC1 for oligodendrocytes ( B , D ). Immunolabelling for Kir4.1 ( E ) and Kir5.1 ( F ) in mice in which EGFP is under the control of the oligodendrocyte-specific Sox10 promoter. G Double immunolabelling for Kir4.1 ( red ) and the oligodenrocyte-specific marker Olig2 ( green ). Insets in Aiv and Civ illustrate negative controls, in the Kir4.1 KO mouse ( Aiv ) and following preincubation with the Kir5.1 blocking peptide ( Civ ). Scale bars 20 μm. Western blot analysis of the brain and optic and nerve for Kir4.1 ( I ) and Kir5.1 ( J ); bands were absent in the negative controls, in the Kir4.1 knock-out mouse ( I ) following preincubation in the Kir5.1 blocking peptide ( J )
    Figure Legend Snippet: Expression of Kir4.1 and Kir5.1 in oligodendrocytes and astrocytes in the cerebellum. Immunolabelling for Kir4.1 and Kir5.1, in combination with GFAP for astrocytes ( A , C ), and APC/CC1 for oligodendrocytes ( B , D ). Immunolabelling for Kir4.1 ( E ) and Kir5.1 ( F ) in mice in which EGFP is under the control of the oligodendrocyte-specific Sox10 promoter. G Double immunolabelling for Kir4.1 ( red ) and the oligodenrocyte-specific marker Olig2 ( green ). Insets in Aiv and Civ illustrate negative controls, in the Kir4.1 KO mouse ( Aiv ) and following preincubation with the Kir5.1 blocking peptide ( Civ ). Scale bars 20 μm. Western blot analysis of the brain and optic and nerve for Kir4.1 ( I ) and Kir5.1 ( J ); bands were absent in the negative controls, in the Kir4.1 knock-out mouse ( I ) following preincubation in the Kir5.1 blocking peptide ( J )

    Techniques Used: Expressing, Mouse Assay, Marker, Blocking Assay, Western Blot, Knock-Out

    Expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Immunolabelling for Kir4.1 ( A , C ) and Kir5.1 ( B , D ), in GFAP-GFP mice to identify astrocytes ( A , B ) and PLP-DsRED mice to identify oligodendrocytes ( C , D ). Cellular expression of Kir4.1 and Kir5.1 is demonstrated by the generation of colocalisation channels ( Av , Bv , Cv , Dv ) from confocal z -stacks ( Aiv , Biv , Civ , Div ), and green and red channels of equal intensity appear yellow . Scale bars 20 μm
    Figure Legend Snippet: Expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Immunolabelling for Kir4.1 ( A , C ) and Kir5.1 ( B , D ), in GFAP-GFP mice to identify astrocytes ( A , B ) and PLP-DsRED mice to identify oligodendrocytes ( C , D ). Cellular expression of Kir4.1 and Kir5.1 is demonstrated by the generation of colocalisation channels ( Av , Bv , Cv , Dv ) from confocal z -stacks ( Aiv , Biv , Civ , Div ), and green and red channels of equal intensity appear yellow . Scale bars 20 μm

    Techniques Used: Expressing, Mouse Assay, Plasmid Purification

    Co-expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Co-immunolocalization of Kir4.1 and Kir5.1 in optic nerve explant cultures, in astrocytes identified by GFAP immunolabelling ( A ) and oligodendrocytes identified by PLP-DsRED ( B ). The overlay and individual channels are illustrated, together with the co-localisation channel for Kir4.1/Kir5.1 ( Aii, Bii ). Boxed areas on overlay images ( Ai , Bi ) are enlarged in Avi – Aviii and Bvi – Bviii , to illustrate punctate colocalization of Kir4.1 and Kir5.1 along processes (some indicated by arrows ). Scale bars 20 μm. Quantification of the number of voxels that were positive for Kir4.1 and Kir5.1 alone and of Kir4.1/Kir5.1 together, in astrocytes ( C , n = 15) and oligodendrocytes ( D , n = 13); data are mean ± SEM. Co-immunoprecipitation of Kir4.1 with Kir5.1 ( E ) and of Kir5.1 with Kir4.1 ( F ) from total brain and optic nerve (ON) lysates; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1, and using the blocking peptide for Kir5.1
    Figure Legend Snippet: Co-expression of Kir4.1 and Kir5.1 in optic nerve oligodendrocytes and astrocytes. Co-immunolocalization of Kir4.1 and Kir5.1 in optic nerve explant cultures, in astrocytes identified by GFAP immunolabelling ( A ) and oligodendrocytes identified by PLP-DsRED ( B ). The overlay and individual channels are illustrated, together with the co-localisation channel for Kir4.1/Kir5.1 ( Aii, Bii ). Boxed areas on overlay images ( Ai , Bi ) are enlarged in Avi – Aviii and Bvi – Bviii , to illustrate punctate colocalization of Kir4.1 and Kir5.1 along processes (some indicated by arrows ). Scale bars 20 μm. Quantification of the number of voxels that were positive for Kir4.1 and Kir5.1 alone and of Kir4.1/Kir5.1 together, in astrocytes ( C , n = 15) and oligodendrocytes ( D , n = 13); data are mean ± SEM. Co-immunoprecipitation of Kir4.1 with Kir5.1 ( E ) and of Kir5.1 with Kir4.1 ( F ) from total brain and optic nerve (ON) lysates; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1, and using the blocking peptide for Kir5.1

    Techniques Used: Expressing, Plasmid Purification, Immunoprecipitation, Knock-Out, Mouse Assay, Blocking Assay

    Plasmalemmal expression of Kir4.1 and Kir5.1 subunit in optic nerve glia. Immunolocalization of Kir4.1 and Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explants of astrocytes identified by GFAP ( A , B ) and oligodendrocytes identified by PLP-DsRed ( C , D ). Scale bars 20 μm. Quantification in astrocytes and oligodendrocytes of total number of voxels immunopositive for Kir4.1 and Kir5.1, compared to voxels that were identified as colocalized for Kir4.1/Na–K-ATPase ( E ) and Kir5.1/Na–K-ATPase ( F ); data are mean ± SEM, n = 13 cells for each analysis. Western blot analysis of Kir5.1 ( G ) and Kir4.1 ( H ) in total optic nerve lysate and plasma membrane fraction. Co-immunoprecipitation of Kir4.1 ( I ) and Kir5.1 ( J ) with PSD95, in total brain and optic nerve (ON) lysate; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1 and preincubation with the blocking peptide for Kir5.1
    Figure Legend Snippet: Plasmalemmal expression of Kir4.1 and Kir5.1 subunit in optic nerve glia. Immunolocalization of Kir4.1 and Kir5.1 with the membrane bound Na–K-ATPase α1 subunit in optic nerve explants of astrocytes identified by GFAP ( A , B ) and oligodendrocytes identified by PLP-DsRed ( C , D ). Scale bars 20 μm. Quantification in astrocytes and oligodendrocytes of total number of voxels immunopositive for Kir4.1 and Kir5.1, compared to voxels that were identified as colocalized for Kir4.1/Na–K-ATPase ( E ) and Kir5.1/Na–K-ATPase ( F ); data are mean ± SEM, n = 13 cells for each analysis. Western blot analysis of Kir5.1 ( G ) and Kir4.1 ( H ) in total optic nerve lysate and plasma membrane fraction. Co-immunoprecipitation of Kir4.1 ( I ) and Kir5.1 ( J ) with PSD95, in total brain and optic nerve (ON) lysate; negative controls were Kir4.1 knock-out mice (−/−) for Kir4.1 and preincubation with the blocking peptide for Kir5.1

    Techniques Used: Expressing, Plasmid Purification, Western Blot, Immunoprecipitation, Knock-Out, Mouse Assay, Blocking Assay

    12) Product Images from "Modulation of the Heteromeric Kir4.1-Kir5.1 Channel by Multiple Neurotransmitters via Gαq-coupled Receptors"

    Article Title: Modulation of the Heteromeric Kir4.1-Kir5.1 Channel by Multiple Neurotransmitters via Gαq-coupled Receptors

    Journal:

    doi: 10.1002/jcp.21169

    Effects of SP on the single channel activity of Kir4.1-Kir5.1
    Figure Legend Snippet: Effects of SP on the single channel activity of Kir4.1-Kir5.1

    Techniques Used: Activity Assay

    Involvement of PKC in the Kir4.1-Kir5.1 inhibition by SP, DOI and TRH
    Figure Legend Snippet: Involvement of PKC in the Kir4.1-Kir5.1 inhibition by SP, DOI and TRH

    Techniques Used: Inhibition

    Expression of Kir4.1-Kir5.1 in brainstem neurons
    Figure Legend Snippet: Expression of Kir4.1-Kir5.1 in brainstem neurons

    Techniques Used: Expressing

    Kir4.1-Kir5.1 channel is inhibited by SP, DOI, and TRH
    Figure Legend Snippet: Kir4.1-Kir5.1 channel is inhibited by SP, DOI, and TRH

    Techniques Used:

    Independent regulation of the Kir4.1-Kir5.1 channel by neurotransmitters and pHi
    Figure Legend Snippet: Independent regulation of the Kir4.1-Kir5.1 channel by neurotransmitters and pHi

    Techniques Used:

    PKC involvement in the modulation of the Kir4.1-Kir5.1 channel by the neurotransmitters
    Figure Legend Snippet: PKC involvement in the modulation of the Kir4.1-Kir5.1 channel by the neurotransmitters

    Techniques Used:

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    Alomone Labs anti kir5 1 kcnj16 antibodies
    Intracellular accumulation of polymyxin B in wild-type, KCNJ15 KO and <t>KCNJ16</t> KO HK-2 cells with the treatment of 25 µM polymyxin B and 50 µM BaCl 2 for 6 h. A Polymyxin B was immunostained with polymyxin <t>antibody</t> and visualized using Alexa Fluor-594 dye (red). The nucleus was counterstained with DAPI (blue). B The plots showing mean fluorescence intensities from each group. The value from control group has been deducted and the mean value from each replicate was plotted ( n = 4)
    Anti Kir5 1 Kcnj16 Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Intracellular accumulation of polymyxin B in wild-type, KCNJ15 KO and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B and 50 µM BaCl 2 for 6 h. A Polymyxin B was immunostained with polymyxin antibody and visualized using Alexa Fluor-594 dye (red). The nucleus was counterstained with DAPI (blue). B The plots showing mean fluorescence intensities from each group. The value from control group has been deducted and the mean value from each replicate was plotted ( n = 4)

    Journal: Cellular and Molecular Life Sciences

    Article Title: Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity

    doi: 10.1007/s00018-022-04316-z

    Figure Lengend Snippet: Intracellular accumulation of polymyxin B in wild-type, KCNJ15 KO and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B and 50 µM BaCl 2 for 6 h. A Polymyxin B was immunostained with polymyxin antibody and visualized using Alexa Fluor-594 dye (red). The nucleus was counterstained with DAPI (blue). B The plots showing mean fluorescence intensities from each group. The value from control group has been deducted and the mean value from each replicate was plotted ( n = 4)

    Article Snippet: Rabbit multiclonal anti-Kir4.2 (KCNJ15) and anti-Kir5.1 (KCNJ16) antibodies were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Fluorescence

    Polymyxin B induced significant electrophysiological changes and membrane depolarization in HK-2 cells. A The resting membrane potential in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 34, 18 and 10, respectively). B Input resistances in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 20, 18 and 10, respectively). C In current clamp mode, polymyxin B induced approximately 30 mV depolarization in WT cells and this was reversible. Depolarization was not induced in KCNJ15 KO cells. Polymyxin B induced membrane potential changes are shown aside ( n = 9, 10 and 7, respectively). D In voltage clamp mode, polymyxin B induced a statistically significant inward current (green) in wild-type HK-2 cells ( n = 8), but not in KCNJ15 KO cells ( n = 8). The current and reversal potential values are shown aside. E Fluorescent signal detection in HK-2 cells with DiBAC, 25 μM polymyxin B, and DiBAC plus 25 μM polymyxin B. F Proportions of DiBAC-positive in wild-type, KCNJ15 KO, and KCNJ16 KO HK-2 cells measured by flow cytometry. G Proportions of DiBAC-positive HK-2 cells in the control and BaCl 2 (10 μM) groups with or without 25 μM polymyxin B treatment measured by flow cytometry ( n = 5 for WT and n = 4 for KOs). Data are shown as box and whisker plots. One-way (for WT) or two-way (for KOs) ANOVA was employed for multi-group comparisons. ** p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity

    doi: 10.1007/s00018-022-04316-z

    Figure Lengend Snippet: Polymyxin B induced significant electrophysiological changes and membrane depolarization in HK-2 cells. A The resting membrane potential in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 34, 18 and 10, respectively). B Input resistances in wild-type, KCNJ15 KO and KCNJ16 KO cells ( n = 20, 18 and 10, respectively). C In current clamp mode, polymyxin B induced approximately 30 mV depolarization in WT cells and this was reversible. Depolarization was not induced in KCNJ15 KO cells. Polymyxin B induced membrane potential changes are shown aside ( n = 9, 10 and 7, respectively). D In voltage clamp mode, polymyxin B induced a statistically significant inward current (green) in wild-type HK-2 cells ( n = 8), but not in KCNJ15 KO cells ( n = 8). The current and reversal potential values are shown aside. E Fluorescent signal detection in HK-2 cells with DiBAC, 25 μM polymyxin B, and DiBAC plus 25 μM polymyxin B. F Proportions of DiBAC-positive in wild-type, KCNJ15 KO, and KCNJ16 KO HK-2 cells measured by flow cytometry. G Proportions of DiBAC-positive HK-2 cells in the control and BaCl 2 (10 μM) groups with or without 25 μM polymyxin B treatment measured by flow cytometry ( n = 5 for WT and n = 4 for KOs). Data are shown as box and whisker plots. One-way (for WT) or two-way (for KOs) ANOVA was employed for multi-group comparisons. ** p

    Article Snippet: Rabbit multiclonal anti-Kir4.2 (KCNJ15) and anti-Kir5.1 (KCNJ16) antibodies were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Flow Cytometry, Whisker Assay

    Knockout or inhibition of Kir4.2 and Kir5.1 prevented polymyxin-induced toxicity in HK-2 cells. A Western blot showing the expression levels of KCNJ15 and KCNJ16 after knockout; actin was used as an internal control. B Viability of wild-type HK-2, KCNJ15 KO and KCNJ16 KO cells following 24-h exposure to 10 and 25 µM polymyxin B ( n = 6). C Viability of HK-2 cells following 24-h exposure to 0–100 µM BaCl 2 with or without 25 µM polymyxin B ( n = 5). D Viability of HK-2 cells following the treatment of 0–25 µM VU0134992 alone or in combination with 25 µM polymyxin B for 24 h ( n = 3 for controls, and n = 4 for treatment groups). E Morphologies of wild-type, KNCJ15 KO, and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B or polymyxin B with the combination of 50 µM BaCl 2 , or 5 µM VU0134992 to wild-type cells . Two-way ANOVA was employed for multi-group comparisons and Tukey's multiple comparison test was employed for post-test. * p

    Journal: Cellular and Molecular Life Sciences

    Article Title: Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity

    doi: 10.1007/s00018-022-04316-z

    Figure Lengend Snippet: Knockout or inhibition of Kir4.2 and Kir5.1 prevented polymyxin-induced toxicity in HK-2 cells. A Western blot showing the expression levels of KCNJ15 and KCNJ16 after knockout; actin was used as an internal control. B Viability of wild-type HK-2, KCNJ15 KO and KCNJ16 KO cells following 24-h exposure to 10 and 25 µM polymyxin B ( n = 6). C Viability of HK-2 cells following 24-h exposure to 0–100 µM BaCl 2 with or without 25 µM polymyxin B ( n = 5). D Viability of HK-2 cells following the treatment of 0–25 µM VU0134992 alone or in combination with 25 µM polymyxin B for 24 h ( n = 3 for controls, and n = 4 for treatment groups). E Morphologies of wild-type, KNCJ15 KO, and KCNJ16 KO HK-2 cells with the treatment of 25 µM polymyxin B or polymyxin B with the combination of 50 µM BaCl 2 , or 5 µM VU0134992 to wild-type cells . Two-way ANOVA was employed for multi-group comparisons and Tukey's multiple comparison test was employed for post-test. * p

    Article Snippet: Rabbit multiclonal anti-Kir4.2 (KCNJ15) and anti-Kir5.1 (KCNJ16) antibodies were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Knock-Out, Inhibition, Western Blot, Expressing

    Viability and progressive motility of spermatozoa was comparable in WT and KO mice. The box plots report the evaluation of viability ( A ) and progressive motility ( B ) for spermatozoa collected from WT and KO mice. Both viability and progressive motility were not significantly changed ( p > 0.05) by KO of Kir5.1 channels.

    Journal: International Journal of Molecular Sciences

    Article Title: Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa

    doi: 10.3390/ijms22115972

    Figure Lengend Snippet: Viability and progressive motility of spermatozoa was comparable in WT and KO mice. The box plots report the evaluation of viability ( A ) and progressive motility ( B ) for spermatozoa collected from WT and KO mice. Both viability and progressive motility were not significantly changed ( p > 0.05) by KO of Kir5.1 channels.

    Article Snippet: Cryostat sections (15 μm) were collected on gelled slides, rinsed in phosphate buffer saline (PBS, pH 7.4), and then incubated overnight in rabbit anti-Kir4.1, anti-Kir4.2, and anti-Kir5.1 antibodies (Alomone labs, Jerusalem, Israel, cat#: APC-035, APC-058, APC-123) diluted 1:1000 in PBS containing 0.3% Triton.

    Techniques: Mouse Assay

    Expression of Kir4.1 and Kir5.1 channels in the cauda epididymis. ( A ) Kir4.1 is expressed in the epithelial cells lining the epididymal ducts (white arrow) and in peritubular smooth muscle of the cauda epididymis (red arrow). Immunoreactivity was absent in the lumen where spermatozoa are located, implying the lack of Kir4.1 expression in these cells. ( B ) Magnified image taken from the upper part of panel A. ( C ) Image showing strong expression of Kir5.1 in spermatozoa. ( D ) Magnification of the lumen of the cauda epididymis. Staining shows the localisation of Kir5.1 subunits in the head of the sperm. Scale bar in ( A ), ( B ), and ( C ) = 25 µm, and in ( D ) = 20 µm.

    Journal: International Journal of Molecular Sciences

    Article Title: Kcnj16 (Kir5.1) Gene Ablation Causes Subfertility and Increases the Prevalence of Morphologically Abnormal Spermatozoa

    doi: 10.3390/ijms22115972

    Figure Lengend Snippet: Expression of Kir4.1 and Kir5.1 channels in the cauda epididymis. ( A ) Kir4.1 is expressed in the epithelial cells lining the epididymal ducts (white arrow) and in peritubular smooth muscle of the cauda epididymis (red arrow). Immunoreactivity was absent in the lumen where spermatozoa are located, implying the lack of Kir4.1 expression in these cells. ( B ) Magnified image taken from the upper part of panel A. ( C ) Image showing strong expression of Kir5.1 in spermatozoa. ( D ) Magnification of the lumen of the cauda epididymis. Staining shows the localisation of Kir5.1 subunits in the head of the sperm. Scale bar in ( A ), ( B ), and ( C ) = 25 µm, and in ( D ) = 20 µm.

    Article Snippet: Cryostat sections (15 μm) were collected on gelled slides, rinsed in phosphate buffer saline (PBS, pH 7.4), and then incubated overnight in rabbit anti-Kir4.1, anti-Kir4.2, and anti-Kir5.1 antibodies (Alomone labs, Jerusalem, Israel, cat#: APC-035, APC-058, APC-123) diluted 1:1000 in PBS containing 0.3% Triton.

    Techniques: Expressing, Staining

    Neurons and glia express Kir7.1 in vitro . (A, B) Cells were isolated from P1‐2 mouse cortex and analysed after 14 days in vitro (DIV) by double immunofluorescence labelling for Kir7.1 (green) and the astrocyte marker GFAP (red, A) or neuronal marker Tuj1 (red, B, asterisks indicate Kir7.1+Tuj1‐ cells that are most likely astrocytes (C‐F) Optic nerve glial explant cultures from P7‐12 mice were analysed at 10DIV by double immunofluorescence labelling for Kir7.1 (green), with the plasmalemmal markers Na + /K + ‐ATPase (C) and PSD95 (D), or the glial Kir channels Kir4.1 (E) and Kir5.1 (F), In all cases, overlays are illustrated (Ai, Bi, Ci, Di, Ei, Fi, co‐expression appears yellow), together with individual channels (Aii‐iii, Bii‐iii, Ci‐iii, Di‐iii, Ei‐iii, Fi‐iii). Scale Bars: A‐B = 50μm; C‐F = 20µm. [Colour figure can be viewed at wileyonlinelibrary.com ]

    Journal: Journal of Anatomy

    Article Title: Glial and neuronal expression of the Inward Rectifying Potassium Channel Kir7.1 in the adult mouse brain

    doi: 10.1111/joa.13048

    Figure Lengend Snippet: Neurons and glia express Kir7.1 in vitro . (A, B) Cells were isolated from P1‐2 mouse cortex and analysed after 14 days in vitro (DIV) by double immunofluorescence labelling for Kir7.1 (green) and the astrocyte marker GFAP (red, A) or neuronal marker Tuj1 (red, B, asterisks indicate Kir7.1+Tuj1‐ cells that are most likely astrocytes (C‐F) Optic nerve glial explant cultures from P7‐12 mice were analysed at 10DIV by double immunofluorescence labelling for Kir7.1 (green), with the plasmalemmal markers Na + /K + ‐ATPase (C) and PSD95 (D), or the glial Kir channels Kir4.1 (E) and Kir5.1 (F), In all cases, overlays are illustrated (Ai, Bi, Ci, Di, Ei, Fi, co‐expression appears yellow), together with individual channels (Aii‐iii, Bii‐iii, Ci‐iii, Di‐iii, Ei‐iii, Fi‐iii). Scale Bars: A‐B = 50μm; C‐F = 20µm. [Colour figure can be viewed at wileyonlinelibrary.com ]

    Article Snippet: Primary antibodies used were rabbit anti‐Kir7.1 at 1 : 300 (Alomone), rat anti‐ myelin basic protein (MBP) at 1 : 300 (Millipore), chicken anti‐GFAP at 1 : 500 (Chemicon), mouse β3‐Tubulin (Tuj1) at 1 : 300 (Millipore), guinea pig anti‐Kir4.1 at 1 : 300 (Alomone), rabbit anti‐Kir5.1 at 1 : 300 (Alomone), mouse anti‐PSD95 at 1 : 500 (Thermo Fisher Scientific) and mouse anti‐Na+ /K+ ATPase α1 at 1 : 500 (Abcam).

    Techniques: In Vitro, Isolation, Immunofluorescence, Marker, Mouse Assay, Expressing