anti kir2 1  (Alomone Labs)


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    Alomone Labs anti kir2 1
    Alterations of protein and mRNA levels of I K1 and I to revealed by Western blot analysis and real-time RT-PCR. (A) Top, examples of Western blot bands of <t>Kir2.1;</t> bottom, quantification of Kir2.1 (B) Top, examples of Western blot bands of Kv4.2; bottom,
    Anti Kir2 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Overexpression of M3 Muscarinic Receptor Is a Novel Strategy for Preventing Sudden Cardiac Death in Transgenic Mice"

    Article Title: Overexpression of M3 Muscarinic Receptor Is a Novel Strategy for Preventing Sudden Cardiac Death in Transgenic Mice

    Journal: Molecular Medicine

    doi: 10.2119/molmed.2011.00093

    Alterations of protein and mRNA levels of I K1 and I to revealed by Western blot analysis and real-time RT-PCR. (A) Top, examples of Western blot bands of Kir2.1; bottom, quantification of Kir2.1 (B) Top, examples of Western blot bands of Kv4.2; bottom,
    Figure Legend Snippet: Alterations of protein and mRNA levels of I K1 and I to revealed by Western blot analysis and real-time RT-PCR. (A) Top, examples of Western blot bands of Kir2.1; bottom, quantification of Kir2.1 (B) Top, examples of Western blot bands of Kv4.2; bottom,

    Techniques Used: Western Blot, Quantitative RT-PCR

    2) Product Images from "Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex"

    Article Title: Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2021.667046

    Localization of inward rectifier potassium channel Kir2.1 in mouse retina. (A) Immunostaining for Kir2.1 (rabbit antibody; red) with DAPI counterstaining (blue) and PSD95 (green) to label photoreceptor terminals. Labels for retinal nuclear layers are as in Figure 1 . Kir2.1 is diffusely present in the IPL, but strongly labeled at the inner limiting membrane (arrowheads) and in the OPL. Five μm confocal stack. (B) Higher magnification view of the OPL reveals Kir2.1 (red) labeling largely below the photoreceptor terminals (green; PSD95 labeling), but with some diffuse and punctate labeling among the terminals. Two μm confocal stack. (Ci) Kir2.1 labeling (guinea pig antibody; red) colocalizes with Calbindin labeled horizontal cells (green) in the OPL. (Cii) Kir2.1 labeling in isolation. (Di) Another view of horizontal cells in the OPL (Calbindin labeling; green) along with mGluR6 labeling (magenta) to show locations of On bipolar cell dendritic tips. Clusters of mGluR6 label indicate cone terminals; three examples are indicated with paired arrowheads. Six μm confocal stack. (Dii) Kir2.1 labeling (rabbit antibody; red) in the same section. (Diii) Merged view of all three labels. Kir2.1 shows both diffuse and punctate labeling in the vicinity of horizontal cell axon terminal projections contacting rods, but little labeling near clusters of dendritic processes contacting cones. (Ei) Higher magnification view of a horizontal cell labeled with Calbindin antibody (green). Several representative axon terminal tips are highlighted with arrowheads. One μm confocal stack. (Eii) Kir2.1 labeling in the same section. (Eiii) Merged view of the two labels shows that tips of horizontal cell axon terminal processes contain punctate clusters of Kir2.1 labeling (arrowheads). (F) Average mRNA expression levels of Kir2.1 (Kcnj2) and kainate receptor subunits GluR6 (Grik2) and GluR7 (Grik3) in mouse retina from single-cell transcriptome data. Kir2.1 is most prominently expressed in horizontal cells and essentially absent from Müller glia. The kainate receptor subunit GluR6 is also found in horizontal cells and Off bipolar cells, but GluR7 is absent from these cell types. Data adapted from Hoang et al. (2020) .
    Figure Legend Snippet: Localization of inward rectifier potassium channel Kir2.1 in mouse retina. (A) Immunostaining for Kir2.1 (rabbit antibody; red) with DAPI counterstaining (blue) and PSD95 (green) to label photoreceptor terminals. Labels for retinal nuclear layers are as in Figure 1 . Kir2.1 is diffusely present in the IPL, but strongly labeled at the inner limiting membrane (arrowheads) and in the OPL. Five μm confocal stack. (B) Higher magnification view of the OPL reveals Kir2.1 (red) labeling largely below the photoreceptor terminals (green; PSD95 labeling), but with some diffuse and punctate labeling among the terminals. Two μm confocal stack. (Ci) Kir2.1 labeling (guinea pig antibody; red) colocalizes with Calbindin labeled horizontal cells (green) in the OPL. (Cii) Kir2.1 labeling in isolation. (Di) Another view of horizontal cells in the OPL (Calbindin labeling; green) along with mGluR6 labeling (magenta) to show locations of On bipolar cell dendritic tips. Clusters of mGluR6 label indicate cone terminals; three examples are indicated with paired arrowheads. Six μm confocal stack. (Dii) Kir2.1 labeling (rabbit antibody; red) in the same section. (Diii) Merged view of all three labels. Kir2.1 shows both diffuse and punctate labeling in the vicinity of horizontal cell axon terminal projections contacting rods, but little labeling near clusters of dendritic processes contacting cones. (Ei) Higher magnification view of a horizontal cell labeled with Calbindin antibody (green). Several representative axon terminal tips are highlighted with arrowheads. One μm confocal stack. (Eii) Kir2.1 labeling in the same section. (Eiii) Merged view of the two labels shows that tips of horizontal cell axon terminal processes contain punctate clusters of Kir2.1 labeling (arrowheads). (F) Average mRNA expression levels of Kir2.1 (Kcnj2) and kainate receptor subunits GluR6 (Grik2) and GluR7 (Grik3) in mouse retina from single-cell transcriptome data. Kir2.1 is most prominently expressed in horizontal cells and essentially absent from Müller glia. The kainate receptor subunit GluR6 is also found in horizontal cells and Off bipolar cells, but GluR7 is absent from these cell types. Data adapted from Hoang et al. (2020) .

    Techniques Used: Immunostaining, Labeling, Isolation, Expressing

    3) Product Images from "Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels, et al. Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels"

    Article Title: Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels, et al. Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15431

    Direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3. (A) Direct interaction between miR‐195 and Cavβ1. A fragment of miR‐195 that binds to CACNB1. miR‐195 is complementary to the CACNB1 gene 943‐949, which encodes Cavβ1, and the corresponding mutant sequence is designed based on the binding site. (B) Luciferase reporter with a CACNB1 fragment capable of binding to miR‐195. The gene was co‐transfected with miR‐195 into HEK293 cells, and miR‐195 reduced the activity of the luciferase reporter gene, ** P
    Figure Legend Snippet: Direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3. (A) Direct interaction between miR‐195 and Cavβ1. A fragment of miR‐195 that binds to CACNB1. miR‐195 is complementary to the CACNB1 gene 943‐949, which encodes Cavβ1, and the corresponding mutant sequence is designed based on the binding site. (B) Luciferase reporter with a CACNB1 fragment capable of binding to miR‐195. The gene was co‐transfected with miR‐195 into HEK293 cells, and miR‐195 reduced the activity of the luciferase reporter gene, ** P

    Techniques Used: Mutagenesis, Sequencing, Binding Assay, Luciferase, Transfection, Activity Assay

    miR‐195 inhibits the expression of Cavβ1, Kir2.1 and Kv4.3 in cardiomyocytes by immunofluorescence and Western blot. (A) Effects of miR‐195 on protein levels of endogenous Cavβ1 in primary cultured cardiomyocytes by Western blot analysis. miR‐195 effectively inhibited the expression of Cavβ1 relative to control group, whereas the scrambled NC miRNA failed to affect the protein levels. In contrast, AMO‐195 rescued the down‐regulation of Cavβ1 elicited by miR‐195. * P
    Figure Legend Snippet: miR‐195 inhibits the expression of Cavβ1, Kir2.1 and Kv4.3 in cardiomyocytes by immunofluorescence and Western blot. (A) Effects of miR‐195 on protein levels of endogenous Cavβ1 in primary cultured cardiomyocytes by Western blot analysis. miR‐195 effectively inhibited the expression of Cavβ1 relative to control group, whereas the scrambled NC miRNA failed to affect the protein levels. In contrast, AMO‐195 rescued the down‐regulation of Cavβ1 elicited by miR‐195. * P

    Techniques Used: Expressing, Immunofluorescence, Western Blot, Cell Culture

    miR‐195 inhibits the protein expression of Cavβ1, Kir2.1 and Kv4.3 in vivo. (A‐C) qPCR showed the changes of CACNB1/KCNJ2/KCND3 transcripts in cardiac tissues from overexpressing miR‐195 mice, n = 5‐9. (D) Verification of the specificity of miR‐195 on Cavβ1. Compared with NC, the expression of Cavβ1 protein in the overexpressed miR‐195 group was decreased. ** P
    Figure Legend Snippet: miR‐195 inhibits the protein expression of Cavβ1, Kir2.1 and Kv4.3 in vivo. (A‐C) qPCR showed the changes of CACNB1/KCNJ2/KCND3 transcripts in cardiac tissues from overexpressing miR‐195 mice, n = 5‐9. (D) Verification of the specificity of miR‐195 on Cavβ1. Compared with NC, the expression of Cavβ1 protein in the overexpressed miR‐195 group was decreased. ** P

    Techniques Used: Expressing, In Vivo, Real-time Polymerase Chain Reaction, Mouse Assay

    4) Product Images from "Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels"

    Article Title: Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvab126

    Ion channels in cardiac MΦ. Current inhibition in the presence of 1 and 10 nM MgTx ( n =15, N =6) ( A and C ) or 3 µM XEN-D0103 ( n =8, N =6) ( B and D ). ( E ) Cumulative inactivation of Type 1 outward currents was abolished after MgTx application. ( F ) Lack of cumulative inactivation was observed in MΦ with Type 2 outward current profile. ( G ) Representative recordings of steady-state inactivation for Types 1 and 2 outward current profiles. ( H ) Steady-state activation and inactivation curves for Type 1 outward currents plotted as a function of holding potential. ( I ) Flow cytometry quantification of cell surface expression of Kv1.3 (in 86.0±3.0% of FACS-purified cardiac MΦ), Kv1.5 (84.2±1.8%) and Kir2.1 (55.9±11.8%) ( N =5); negative control data are from same cells and labels, omitting the primary antibody (
    Figure Legend Snippet: Ion channels in cardiac MΦ. Current inhibition in the presence of 1 and 10 nM MgTx ( n =15, N =6) ( A and C ) or 3 µM XEN-D0103 ( n =8, N =6) ( B and D ). ( E ) Cumulative inactivation of Type 1 outward currents was abolished after MgTx application. ( F ) Lack of cumulative inactivation was observed in MΦ with Type 2 outward current profile. ( G ) Representative recordings of steady-state inactivation for Types 1 and 2 outward current profiles. ( H ) Steady-state activation and inactivation curves for Type 1 outward currents plotted as a function of holding potential. ( I ) Flow cytometry quantification of cell surface expression of Kv1.3 (in 86.0±3.0% of FACS-purified cardiac MΦ), Kv1.5 (84.2±1.8%) and Kir2.1 (55.9±11.8%) ( N =5); negative control data are from same cells and labels, omitting the primary antibody (

    Techniques Used: Inhibition, Activation Assay, Flow Cytometry, Expressing, FACS, Purification, Negative Control

    Deconstruction of simulated currents. ( A ) Cardiac MΦ with Type 1 or 2 outward current profiles in the presence of an inward-rectifier current can be reproduced by a background current ( I b ), an outward current ( I outward, I Kv1.3 for Type 1, left, or I Kv1.5 for Type 2, right) and an inward-rectifier current ( I Kir2.1 ). Comparison between simulated (red lines) and experimentally recorded currents (black traces). ( B ) Deconstruction of the different simulated currents. ( C ) Simulated IV curve after adding the three currents represented in ( B ).
    Figure Legend Snippet: Deconstruction of simulated currents. ( A ) Cardiac MΦ with Type 1 or 2 outward current profiles in the presence of an inward-rectifier current can be reproduced by a background current ( I b ), an outward current ( I outward, I Kv1.3 for Type 1, left, or I Kv1.5 for Type 2, right) and an inward-rectifier current ( I Kir2.1 ). Comparison between simulated (red lines) and experimentally recorded currents (black traces). ( B ) Deconstruction of the different simulated currents. ( C ) Simulated IV curve after adding the three currents represented in ( B ).

    Techniques Used:

    5) Product Images from "Tbx20 controls the expression of the KCNH2 gene and of hERG channels"

    Article Title: Tbx20 controls the expression of the KCNH2 gene and of hERG channels

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1612383114

    Western blot images and their corresponding total protein gels showing Kv7.1, minK, Kir2.1, and Cav1.2 expression (arrows) in HL-1 cells transfected or not with Tbx20 WT or p.R311C. As depicted in A and C , the presence of any form of Tbx20 did not modify
    Figure Legend Snippet: Western blot images and their corresponding total protein gels showing Kv7.1, minK, Kir2.1, and Cav1.2 expression (arrows) in HL-1 cells transfected or not with Tbx20 WT or p.R311C. As depicted in A and C , the presence of any form of Tbx20 did not modify

    Techniques Used: Western Blot, Expressing, Transfection

    6) Product Images from "Activity-dependent refinement of nasal retinal projections drives topographic map sharpening in the teleost visual system"

    Article Title: Activity-dependent refinement of nasal retinal projections drives topographic map sharpening in the teleost visual system

    Journal: bioRxiv

    doi: 10.1101/2020.12.14.422653

    Blocking neuronal activity in RGCs prevents the sharpening of the antero-posterior retinotopic map . ( A-F” ) Development of the antero-posterior retinotopic map in Kir2.1 (A-B”), KirMUT (C-D”), and uninjected embryos (E-F”) from 4 to 5 dpf. ( A-F ) EGFP-positive nasal axons innervate the posterior half of the tectum in all three experimental groups. The area covered by nasal axons in the anterior half of the tectum appears to decrease between 4 and 5 dpf in KirMut and uninjected embryos (arrows) but does not seem to change in Kir2.1 embryos. ( A’-F’ ) TagRFP-positive temporal axons specifically target the anterior half of the tectum. Confocal microscopy, scale bar: 50 µm. ( G-H ) Mean fluorescence intensities of EGFP and TagRFP were normalized to their respective maximum value and plotted along the antero-posterior axis of the tectum. The boundary sharpness between the temporal and nasal arborization domains (shown as the distance between EGFP 50% and TagRFP 50% ) does not change between 4 and 5 dpf in Kir2.1 embryos. ( I-L ) The boundary sharpness between the temporal and nasal arborization domains decreases between 4 and 5 dpf in KirMUT (I, J) and uninjected (K, L) embryos, indicating the formation of a more precise antero-posterior retinotopic map. Data represent mean ± SEM. n = 15 Kir2.1 embryos, 12 KirMut embryos, 15 uninjected embryos.
    Figure Legend Snippet: Blocking neuronal activity in RGCs prevents the sharpening of the antero-posterior retinotopic map . ( A-F” ) Development of the antero-posterior retinotopic map in Kir2.1 (A-B”), KirMUT (C-D”), and uninjected embryos (E-F”) from 4 to 5 dpf. ( A-F ) EGFP-positive nasal axons innervate the posterior half of the tectum in all three experimental groups. The area covered by nasal axons in the anterior half of the tectum appears to decrease between 4 and 5 dpf in KirMut and uninjected embryos (arrows) but does not seem to change in Kir2.1 embryos. ( A’-F’ ) TagRFP-positive temporal axons specifically target the anterior half of the tectum. Confocal microscopy, scale bar: 50 µm. ( G-H ) Mean fluorescence intensities of EGFP and TagRFP were normalized to their respective maximum value and plotted along the antero-posterior axis of the tectum. The boundary sharpness between the temporal and nasal arborization domains (shown as the distance between EGFP 50% and TagRFP 50% ) does not change between 4 and 5 dpf in Kir2.1 embryos. ( I-L ) The boundary sharpness between the temporal and nasal arborization domains decreases between 4 and 5 dpf in KirMUT (I, J) and uninjected (K, L) embryos, indicating the formation of a more precise antero-posterior retinotopic map. Data represent mean ± SEM. n = 15 Kir2.1 embryos, 12 KirMut embryos, 15 uninjected embryos.

    Techniques Used: Blocking Assay, Activity Assay, Confocal Microscopy, Fluorescence

    Blocking neuronal activity in RGCs prevents the refinement of nasal projections . ( A ) Like uninjected control embryos, embryos expressing Kir2.1 or KirMut in RGCs (thereafter referred to as Kir2.1 or KirMUT embryos) display an increase in total tectal coverage between 4 and 5 dpf. ( B ) The Temporal Arborization Field index is similar in Kir2.1, KirMUT and uninjected embryos at 4 and 5 dpf. ( C ) The Equatorial Alignment Index does not significantly differ between days or between experimental groups. ( D ) The area covered by EGFP-positive nasal axons in the posterior half of the tectum significantly increases between 4 and 5 dpf in Kir2.1 embryos but remains significantly smaller than that in the uninjected controls at both time points. ( E ) The anterior tectal area covered by EGFP-positive nasal axons slightly increases between 4 and 5 dpf in Kir2.1 embryos while it decreases in KirMut and uninjected embryos, suggesting a lack of refinement of the nasal projections in Kir2.1 embryos. ( F ) While the Nasal Axon Mis- targeting Index significantly decreases between 4 and 5 dpf in KirMut and uninjected embryos, it remains stable in Kir2.1 embryos, indicating a lack of refinement of the nasal projections. ( G ) While the Refinement Index between 4 and 5 dpf is greater than 1 in KirMut and uninjected embryos, it averages 1 in Kir2.1 embryos, confirming the absence of refinement of the nasal projection domain. ( H ) The Boundary Sharpness Index significantly decreases in KirMut and uninjected embryos but remains stable in Kir2.1 embryos, indicating that the boundary between the EGFP and TagRFP domains does not refine over time in embryos expressing Kir2.1 in RGCs. ( A-H ) Data represent mean ± SEM. n = 15 Kir2.1 embryos, 12 KirMut embryos, 15 uninjected embryos. Four biological replicates representing independent experiments are color-coded in grey, teal, purple and orange, respectively. Statistical Analysis: (A-F, H) mixed-effects one-way ANOVA with Tukey’s posthoc test; (G) paired t-test compared to a control of 1 (1 representing no change); *p
    Figure Legend Snippet: Blocking neuronal activity in RGCs prevents the refinement of nasal projections . ( A ) Like uninjected control embryos, embryos expressing Kir2.1 or KirMut in RGCs (thereafter referred to as Kir2.1 or KirMUT embryos) display an increase in total tectal coverage between 4 and 5 dpf. ( B ) The Temporal Arborization Field index is similar in Kir2.1, KirMUT and uninjected embryos at 4 and 5 dpf. ( C ) The Equatorial Alignment Index does not significantly differ between days or between experimental groups. ( D ) The area covered by EGFP-positive nasal axons in the posterior half of the tectum significantly increases between 4 and 5 dpf in Kir2.1 embryos but remains significantly smaller than that in the uninjected controls at both time points. ( E ) The anterior tectal area covered by EGFP-positive nasal axons slightly increases between 4 and 5 dpf in Kir2.1 embryos while it decreases in KirMut and uninjected embryos, suggesting a lack of refinement of the nasal projections in Kir2.1 embryos. ( F ) While the Nasal Axon Mis- targeting Index significantly decreases between 4 and 5 dpf in KirMut and uninjected embryos, it remains stable in Kir2.1 embryos, indicating a lack of refinement of the nasal projections. ( G ) While the Refinement Index between 4 and 5 dpf is greater than 1 in KirMut and uninjected embryos, it averages 1 in Kir2.1 embryos, confirming the absence of refinement of the nasal projection domain. ( H ) The Boundary Sharpness Index significantly decreases in KirMut and uninjected embryos but remains stable in Kir2.1 embryos, indicating that the boundary between the EGFP and TagRFP domains does not refine over time in embryos expressing Kir2.1 in RGCs. ( A-H ) Data represent mean ± SEM. n = 15 Kir2.1 embryos, 12 KirMut embryos, 15 uninjected embryos. Four biological replicates representing independent experiments are color-coded in grey, teal, purple and orange, respectively. Statistical Analysis: (A-F, H) mixed-effects one-way ANOVA with Tukey’s posthoc test; (G) paired t-test compared to a control of 1 (1 representing no change); *p

    Techniques Used: Blocking Assay, Activity Assay, Expressing

    Expressing Kir2.1 in RGCs blocks larvae’s visually mediated background adaptation . ( A ) Experimental work-flow to assess the functionality of Kir2.1 transgene in injected embryos. A UAS:Kir2.1-2A-mKate2CAAX or a UAS:Kir2.1MUT-2A-mKate2CAAX transgene was injected in zygotes from an [ isl2b:gal4 ] outcross at one-cell stage. Embryos were divided into two groups: one raised in the absence of PTU for conducting a Visually-mediated Background Adaptation (VBA) assay at 5 dpf, and another raised with PTU for the analysis of mKate2 expression at 4 dpf by confocal microscopy. Based on the VBA assay, embryos were sorted into four groups: embryos expressing Kir2.1 that had expanded pigmentation (“large melanophores”) despite bright illumination, embryos expressing Kir2.1 that had “smaller melanophores”, embryos expressing KirMut, and uninjected embryos as another control group. After confocal imaging at 4 dpf, embryos were sorted into three categories based on the intensity of mKate2 expression: embryos expressing Kir2.1 that had high mKate2 expression (see Material and Methods for high mKate2 expression definition), embryos expressing Kir2.1 that had low mKate2 expression, and embryos expressing KirMut. All embryos were then used for analyzing Kir2.1/Kir2.1MUT and mKate2 expression by Western blot at 5 dpf. ( B-E’ ) VBA in embryos expressing Kir2.1 or KirMut in RGCs (thereafter referred to as Kir2.1 or KirMUT embryos) and uninjected embryos. Pictures of embryos in a dorsal view (B, C, D, and E) were binarized using a threshold of 40 (B’, C’, D’ and E’). The area covered by pigmented melanophores was measured in a region caudal to the eyes and rostral to the medulla oblongata (orange box). ( B-C’ ) Kir2.1 embryos demonstrated two levels of dark, expanded pigmentation despite bright illumination. ( B, B’ ) Some Kir2.1 embryos retained dispersed melanosomes (“large melanophores”) in response to light, indicating a lack of VBA. ( C, C’ ) Other Kir2.1 embryos had more restricted but yet abnormally expanded melanin in response to light (“smaller melanophores”), indicating a reduced VBA. ( D-E’ ) In contrast to Kir2.1 embryos, KirMut and uninjected embryos showed fully aggregated melanin in response to bright illumination. ( F ) The area covered by pigmented melanophores (pigmentation measured in the region delineated by orange boxes in B’-E’) is significantly larger in Kir2.1 embryos with large or smaller melanophores than in KirMUT or uninjected embryos. Statistical analysis: One-way ANOVA with Tukey’s posthoc test; *p
    Figure Legend Snippet: Expressing Kir2.1 in RGCs blocks larvae’s visually mediated background adaptation . ( A ) Experimental work-flow to assess the functionality of Kir2.1 transgene in injected embryos. A UAS:Kir2.1-2A-mKate2CAAX or a UAS:Kir2.1MUT-2A-mKate2CAAX transgene was injected in zygotes from an [ isl2b:gal4 ] outcross at one-cell stage. Embryos were divided into two groups: one raised in the absence of PTU for conducting a Visually-mediated Background Adaptation (VBA) assay at 5 dpf, and another raised with PTU for the analysis of mKate2 expression at 4 dpf by confocal microscopy. Based on the VBA assay, embryos were sorted into four groups: embryos expressing Kir2.1 that had expanded pigmentation (“large melanophores”) despite bright illumination, embryos expressing Kir2.1 that had “smaller melanophores”, embryos expressing KirMut, and uninjected embryos as another control group. After confocal imaging at 4 dpf, embryos were sorted into three categories based on the intensity of mKate2 expression: embryos expressing Kir2.1 that had high mKate2 expression (see Material and Methods for high mKate2 expression definition), embryos expressing Kir2.1 that had low mKate2 expression, and embryos expressing KirMut. All embryos were then used for analyzing Kir2.1/Kir2.1MUT and mKate2 expression by Western blot at 5 dpf. ( B-E’ ) VBA in embryos expressing Kir2.1 or KirMut in RGCs (thereafter referred to as Kir2.1 or KirMUT embryos) and uninjected embryos. Pictures of embryos in a dorsal view (B, C, D, and E) were binarized using a threshold of 40 (B’, C’, D’ and E’). The area covered by pigmented melanophores was measured in a region caudal to the eyes and rostral to the medulla oblongata (orange box). ( B-C’ ) Kir2.1 embryos demonstrated two levels of dark, expanded pigmentation despite bright illumination. ( B, B’ ) Some Kir2.1 embryos retained dispersed melanosomes (“large melanophores”) in response to light, indicating a lack of VBA. ( C, C’ ) Other Kir2.1 embryos had more restricted but yet abnormally expanded melanin in response to light (“smaller melanophores”), indicating a reduced VBA. ( D-E’ ) In contrast to Kir2.1 embryos, KirMut and uninjected embryos showed fully aggregated melanin in response to bright illumination. ( F ) The area covered by pigmented melanophores (pigmentation measured in the region delineated by orange boxes in B’-E’) is significantly larger in Kir2.1 embryos with large or smaller melanophores than in KirMUT or uninjected embryos. Statistical analysis: One-way ANOVA with Tukey’s posthoc test; *p

    Techniques Used: Expressing, Injection, Confocal Microscopy, Imaging, Western Blot

    7) Product Images from "Kir2.4 and Kir2.1 K+ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity"

    Article Title: Kir2.4 and Kir2.1 K+ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2002.026047

    Western blots of cell lysates from Cos-7 cells transfected with Kir2.1-His6, Kir2.4-FLAG or both The constructs transfected and antibody probes used are indicated at the bottom. + indicates construct transfected, – indicates construct not present. Primary antibodies are designated by F for anti-FLAG, 2.1 for anti-Kir2.1 and H for anti-His. Arrows on the left indicate molecular masses of specific bands detected by specific primary antibodies. Molecular masses are given in kilodaltons (kDa). Lane 1 is a Western blot of Cos-7 cell lysate transfected with Kir2.4-FLAG and purified on a FLAG-Affinity gel (Sigma Chemical Co.). Cell lysates in lanes 2 to 7 were subjected to His 6 -pulldown prior to incubation with the primary antibody. Lanes 2 and 3 were obtained with a lysate of cells transfected with Kir2.1-His 6 only and lane 4 was obtained with a lysate of cells co-transfected with Kir2.4-FLAG and Kir2.1-His 6 . Lane 5 was obtained from cells transfected with Kir2.1-His 6 and incubated with anti-FLAG after His-pulldown. Lane 6 is a Western blot of lysate of Cos-7 cells transfected with Kir2.4-FLAG only incubated with anti-FLAG. Lane 7 is a Western blot with crude (non-transfected) Cos-7 cell lysate incubated with anti-Kir2.1.
    Figure Legend Snippet: Western blots of cell lysates from Cos-7 cells transfected with Kir2.1-His6, Kir2.4-FLAG or both The constructs transfected and antibody probes used are indicated at the bottom. + indicates construct transfected, – indicates construct not present. Primary antibodies are designated by F for anti-FLAG, 2.1 for anti-Kir2.1 and H for anti-His. Arrows on the left indicate molecular masses of specific bands detected by specific primary antibodies. Molecular masses are given in kilodaltons (kDa). Lane 1 is a Western blot of Cos-7 cell lysate transfected with Kir2.4-FLAG and purified on a FLAG-Affinity gel (Sigma Chemical Co.). Cell lysates in lanes 2 to 7 were subjected to His 6 -pulldown prior to incubation with the primary antibody. Lanes 2 and 3 were obtained with a lysate of cells transfected with Kir2.1-His 6 only and lane 4 was obtained with a lysate of cells co-transfected with Kir2.4-FLAG and Kir2.1-His 6 . Lane 5 was obtained from cells transfected with Kir2.1-His 6 and incubated with anti-FLAG after His-pulldown. Lane 6 is a Western blot of lysate of Cos-7 cells transfected with Kir2.4-FLAG only incubated with anti-FLAG. Lane 7 is a Western blot with crude (non-transfected) Cos-7 cell lysate incubated with anti-Kir2.1.

    Techniques Used: Western Blot, Transfection, Construct, Purification, Incubation

    8) Product Images from "Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration"

    Article Title: Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22042081

    The experimental steps showing the inhibitory effect of potassium channels on microglial migration. ( A ) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. ( B ) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.
    Figure Legend Snippet: The experimental steps showing the inhibitory effect of potassium channels on microglial migration. ( A ) The monolayer of BV2 microglial cells was scratched with a sterile pipette tip and incubated with the medium or the pharmacological inhibitors or the cells were transfected with small interfering RNA (siRNA); pictures were analyzed at t24h and the inhibition of Kv1.3 and Kir2.1 reduced the rate of migration in the BV2 microglial cell line. ( B ) CX3CR1-eGFP transgenic mice were subjected to SNI or sham surgeries, the ipsilateral spinal cord dorsal horn (SC-DH) was dissected and cultured for 3 h in 8 µm pore inserts, in the culture medium or in the presence of the inhibitors, and the rate of migration was quantified in each condition. Different mechanisms by which the inhibition of the investigated potassium channels may influence the microglial migration rate, are proposed in the outlined box. PBS: phosphate buffer saline; PFA: paraformaldehyde.

    Techniques Used: Migration, Transferring, Incubation, Transfection, Small Interfering RNA, Inhibition, Transgenic Assay, Mouse Assay, Cell Culture

    Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 ( A ), Kv1.5 ( B ), Kir2.1 ( C ) and the negative control with Alexa 568 ( D ). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.
    Figure Legend Snippet: Immunolabeling of potassium channels in cultured BV2 microglial cells. The cells were stained with antibodies targeting Kv1.3 ( A ), Kv1.5 ( B ), Kir2.1 ( C ) and the negative control with Alexa 568 ( D ). The coverslips were mounted using the Prolong antifade with DAPI in blue. The channel was pseudo-colored in green for improved visualization. The images are representative of three independent experiments. Scale bar: 40 µm.

    Techniques Used: Immunolabeling, Cell Culture, Staining, Negative Control

    The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration in control conditions ( E ) and after the inhibitor at t24h ( F – H ). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 ( B , D ), whereas blocking Kv1.5 has no effect on cellular migration ( C ). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p
    Figure Legend Snippet: The contribution of potassium channels in BV2 microglial migration. The representative images show the robust and repetitive scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration in control conditions ( E ) and after the inhibitor at t24h ( F – H ). The histograms show that BV2 microglial cells migrate less after the inhibition of Kv1.3 and Kir2.1 ( B , D ), whereas blocking Kv1.5 has no effect on cellular migration ( C ). Scale bar: 100 µm. All the statistical analysis is represented as Mean ± SD, ***: p

    Techniques Used: Migration, Transferring, Inhibition, Blocking Assay

    Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV ( A – C ), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD ( n = 3 independent experiments).
    Figure Legend Snippet: Electrophysiological properties of BV2 cells. For all Kv1.3, Kv1.5 and Kir2.1 potassium channels, the electrophysiological characteristics are represented by the intensity/voltage (I/V) curves extracted from the step recordings, representing the total current elicited by a voltage protocol starting from −120 mV, in 10 mV increments, to +40 mV ( A – C ), in control conditions and after each specific inhibitor. The inhibition effect of the of each blocker can be seen from the I/V curves. The insets represent the comparison of the current amplitude elicited by a voltage step at −160 mV and +40 mV. All the data are represented as Mean ± SD ( n = 3 independent experiments).

    Techniques Used: Inhibition

    BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration after scrambled and silencing siRNA at t24h ( B – D ) and at t48h ( E – G ). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h ( H ) and at t48h ( I ). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p
    Figure Legend Snippet: BV2 microglial migration after the silencing of the Kv1.3 and Kir2.1 channels. The representative images show the scratch made with the 200 µL sterile pipette tip at t0h ( A ) and the cell migration after scrambled and silencing siRNA at t24h ( B – D ) and at t48h ( E – G ). The bar graphs show the BV2 migration after scrambled and silencing siRNA at t24h ( H ) and at t48h ( I ). Scale bar: 100 µm. All the statistical analysis are represented as Mean ± SD, ***: p

    Techniques Used: Migration, Transferring

    The migration of primary microglial cells through inserts with 8 µm pores. ( A ) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham ( B – D ) and SNI conditions ( E – G ). All the statistical analysis is represented as Mean ± SD, *: p
    Figure Legend Snippet: The migration of primary microglial cells through inserts with 8 µm pores. ( A ) The migration rate of primary microglial cells after the spared nerve injury (SNI) surgery is reduced compared with sham conditions. Histograms showing the contribution of each potassium channel, Kv1.3, Kv1.5 and Kir2.1, to microglial migration, in both sham ( B – D ) and SNI conditions ( E – G ). All the statistical analysis is represented as Mean ± SD, *: p

    Techniques Used: Migration

    9) Product Images from "Kir2.4 and Kir2.1 K+ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity"

    Article Title: Kir2.4 and Kir2.1 K+ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2002.026047

    Western blots of cell lysates from Cos-7 cells transfected with Kir2.1-His6, Kir2.4-FLAG or both The constructs transfected and antibody probes used are indicated at the bottom. + indicates construct transfected, – indicates construct not present. Primary antibodies are designated by F for anti-FLAG, 2.1 for anti-Kir2.1 and H for anti-His. Arrows on the left indicate molecular masses of specific bands detected by specific primary antibodies. Molecular masses are given in kilodaltons (kDa). Lane 1 is a Western blot of Cos-7 cell lysate transfected with Kir2.4-FLAG and purified on a FLAG-Affinity gel (Sigma Chemical Co.). Cell lysates in lanes 2 to 7 were subjected to His 6 -pulldown prior to incubation with the primary antibody. Lanes 2 and 3 were obtained with a lysate of cells transfected with Kir2.1-His 6 only and lane 4 was obtained with a lysate of cells co-transfected with Kir2.4-FLAG and Kir2.1-His 6 . Lane 5 was obtained from cells transfected with Kir2.1-His 6 and incubated with anti-FLAG after His-pulldown. Lane 6 is a Western blot of lysate of Cos-7 cells transfected with Kir2.4-FLAG only incubated with anti-FLAG. Lane 7 is a Western blot with crude (non-transfected) Cos-7 cell lysate incubated with anti-Kir2.1.
    Figure Legend Snippet: Western blots of cell lysates from Cos-7 cells transfected with Kir2.1-His6, Kir2.4-FLAG or both The constructs transfected and antibody probes used are indicated at the bottom. + indicates construct transfected, – indicates construct not present. Primary antibodies are designated by F for anti-FLAG, 2.1 for anti-Kir2.1 and H for anti-His. Arrows on the left indicate molecular masses of specific bands detected by specific primary antibodies. Molecular masses are given in kilodaltons (kDa). Lane 1 is a Western blot of Cos-7 cell lysate transfected with Kir2.4-FLAG and purified on a FLAG-Affinity gel (Sigma Chemical Co.). Cell lysates in lanes 2 to 7 were subjected to His 6 -pulldown prior to incubation with the primary antibody. Lanes 2 and 3 were obtained with a lysate of cells transfected with Kir2.1-His 6 only and lane 4 was obtained with a lysate of cells co-transfected with Kir2.4-FLAG and Kir2.1-His 6 . Lane 5 was obtained from cells transfected with Kir2.1-His 6 and incubated with anti-FLAG after His-pulldown. Lane 6 is a Western blot of lysate of Cos-7 cells transfected with Kir2.4-FLAG only incubated with anti-FLAG. Lane 7 is a Western blot with crude (non-transfected) Cos-7 cell lysate incubated with anti-Kir2.1.

    Techniques Used: Western Blot, Transfection, Construct, Purification, Incubation

    10) Product Images from "Pressure-dependent modulation of inward-rectifying K+ channels: implications for cation homeostasis and K+ dynamics in glaucoma"

    Article Title: Pressure-dependent modulation of inward-rectifying K+ channels: implications for cation homeostasis and K+ dynamics in glaucoma

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00444.2018

    Intensity of inwardly rectifying K + (Kir) channel staining in Müller glia cultures following pressure elevation. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 48 h. A : representative fluorescent micrographs of Müller glia from each condition. Immunolabeling of Kir2.1 (green), Kir4.1 (green), and CD44 (red) was performed. Panels directly below the fluoromicrographs are a z -plane view of the image, with associated plot of fluorescent intensity. B : bar graphs of the mean intensity of Kir2.1 and Kir4.1 staining compared between ambient and elevated pressure. Kir2.1: n (Amb) = 29 cells; n (Elev) = 18; Kir4.1: n (Amb) = 19; n (Elev) = 20. Images taken at ×60; scale bar = 40 µm. Inset taken at ×60 + ×2.5 zoom; scale bar = 10 µm. Student’s t -test was used to analyze statistical significance. ** P
    Figure Legend Snippet: Intensity of inwardly rectifying K + (Kir) channel staining in Müller glia cultures following pressure elevation. Primary, purified cultures of Müller glia were exposed to ambient (Amb) or elevated (Elev) pressure for 48 h. A : representative fluorescent micrographs of Müller glia from each condition. Immunolabeling of Kir2.1 (green), Kir4.1 (green), and CD44 (red) was performed. Panels directly below the fluoromicrographs are a z -plane view of the image, with associated plot of fluorescent intensity. B : bar graphs of the mean intensity of Kir2.1 and Kir4.1 staining compared between ambient and elevated pressure. Kir2.1: n (Amb) = 29 cells; n (Elev) = 18; Kir4.1: n (Amb) = 19; n (Elev) = 20. Images taken at ×60; scale bar = 40 µm. Inset taken at ×60 + ×2.5 zoom; scale bar = 10 µm. Student’s t -test was used to analyze statistical significance. ** P

    Techniques Used: Staining, Purification, Immunolabeling

    Intensity of inwardly rectifying K + (Kir) channel staining in glaucomatous retinal sections. A : representative fluorescent micrographs of retinal sections from saline- or microbead-injected eyes. Immunolabeling of Kir2.1 (green), Kir4.1 (green), and CD44 (red) was performed. B : bar graphs of the mean intensity of Kir2.1 and Kir4.1 staining compared between saline and microbead retina. Kir2.1: n (PBS)= 7 retina sections; n (MOM)= 7; Kir4.1: n (PBS)= 6; n (MOM) = 7. Images taken at ×40; scale bar = 20 µm. Student’s t -test was used to analyze statistical significance. * P
    Figure Legend Snippet: Intensity of inwardly rectifying K + (Kir) channel staining in glaucomatous retinal sections. A : representative fluorescent micrographs of retinal sections from saline- or microbead-injected eyes. Immunolabeling of Kir2.1 (green), Kir4.1 (green), and CD44 (red) was performed. B : bar graphs of the mean intensity of Kir2.1 and Kir4.1 staining compared between saline and microbead retina. Kir2.1: n (PBS)= 7 retina sections; n (MOM)= 7; Kir4.1: n (PBS)= 6; n (MOM) = 7. Images taken at ×40; scale bar = 20 µm. Student’s t -test was used to analyze statistical significance. * P

    Techniques Used: Staining, Injection, Immunolabeling

    11) Product Images from "SDF-1α and LPA Modulate Microglia Potassium Channels Through Rho GTPases to Regulate Cell Morphology"

    Article Title: SDF-1α and LPA Modulate Microglia Potassium Channels Through Rho GTPases to Regulate Cell Morphology

    Journal: Glia

    doi: 10.1002/glia.22543

    SDF-1 α signaling involves PI3-kinase and the small GTPase Rac. A : Left panels show a control untreated microglia, the middle panels shows SDF-1 α (100 nM) treated microglia and the right panels show microglia treated with SDF-1 α (100 nM) and wortmannin (50 nM). The top panels show actin stained red with Alexafluor 594-conjugated phalloidin; middle panel shows the microglia marker in green with FITC-conjugated F4/80 antibody and bottom panel shows an overlay image with yellow showing areas of colocalization. B : Percent change in Kir2.1-like current amplitude after exposure to SDF-1 α (100 ng/mL). Bath application of wortmannin (50 nM) or expression of dominant-negative Rac (17N) prevented stimulation of the current by SDF-1 α . C : Time course of change in normalized peak current in control cells (control, black, n = 6) and cells dialyzed with 6 μg/mL constitutively active Rac1 protein (61L, green, n = 6) or constitutively active RhoA protein (Rho 63L, red, n = 5) from the patch-pipette into the cell. The currents were normalized to the current amplitude at the start of the experiments to allow comparison between cells. * P
    Figure Legend Snippet: SDF-1 α signaling involves PI3-kinase and the small GTPase Rac. A : Left panels show a control untreated microglia, the middle panels shows SDF-1 α (100 nM) treated microglia and the right panels show microglia treated with SDF-1 α (100 nM) and wortmannin (50 nM). The top panels show actin stained red with Alexafluor 594-conjugated phalloidin; middle panel shows the microglia marker in green with FITC-conjugated F4/80 antibody and bottom panel shows an overlay image with yellow showing areas of colocalization. B : Percent change in Kir2.1-like current amplitude after exposure to SDF-1 α (100 ng/mL). Bath application of wortmannin (50 nM) or expression of dominant-negative Rac (17N) prevented stimulation of the current by SDF-1 α . C : Time course of change in normalized peak current in control cells (control, black, n = 6) and cells dialyzed with 6 μg/mL constitutively active Rac1 protein (61L, green, n = 6) or constitutively active RhoA protein (Rho 63L, red, n = 5) from the patch-pipette into the cell. The currents were normalized to the current amplitude at the start of the experiments to allow comparison between cells. * P

    Techniques Used: Staining, Marker, Expressing, Dominant Negative Mutation, Transferring

    12) Product Images from "Electrical and histological remodeling of the pulmonary vein in 2K1C hypertensive rats: Indication of initiation and maintenance of atrial fibrillation"

    Article Title: Electrical and histological remodeling of the pulmonary vein in 2K1C hypertensive rats: Indication of initiation and maintenance of atrial fibrillation

    Journal: Anatolian Journal of Cardiology

    doi: 10.14744/AnatolJCardiol.2017.7844

    Protein levels of the ion channel components Na v 1.5, Kir2.1, Kir2.3, and Ca v 1.2, analyzed by western blot and normalized to GAPDH Data are expressed as mean±SD. *p
    Figure Legend Snippet: Protein levels of the ion channel components Na v 1.5, Kir2.1, Kir2.3, and Ca v 1.2, analyzed by western blot and normalized to GAPDH Data are expressed as mean±SD. *p

    Techniques Used: Western Blot

    13) Product Images from "Kir2.4 and Kir2.1 K+ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity"

    Article Title: Kir2.4 and Kir2.1 K+ channel subunits co-assemble: a potential new contributor to inward rectifier current heterogeneity

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2002.026047

    Western blots of cell lysates from Cos-7 cells transfected with Kir2.1-His6, Kir2.4-FLAG or both The constructs transfected and antibody probes used are indicated at the bottom. + indicates construct transfected, – indicates construct not present. Primary antibodies are designated by F for anti-FLAG, 2.1 for anti-Kir2.1 and H for anti-His. Arrows on the left indicate molecular masses of specific bands detected by specific primary antibodies. Molecular masses are given in kilodaltons (kDa). Lane 1 is a Western blot of Cos-7 cell lysate transfected with Kir2.4-FLAG and purified on a FLAG-Affinity gel (Sigma Chemical Co.). Cell lysates in lanes 2 to 7 were subjected to His 6 -pulldown prior to incubation with the primary antibody. Lanes 2 and 3 were obtained with a lysate of cells transfected with Kir2.1-His 6 only and lane 4 was obtained with a lysate of cells co-transfected with Kir2.4-FLAG and Kir2.1-His 6 . Lane 5 was obtained from cells transfected with Kir2.1-His 6 and incubated with anti-FLAG after His-pulldown. Lane 6 is a Western blot of lysate of Cos-7 cells transfected with Kir2.4-FLAG only incubated with anti-FLAG. Lane 7 is a Western blot with crude (non-transfected) Cos-7 cell lysate incubated with anti-Kir2.1.
    Figure Legend Snippet: Western blots of cell lysates from Cos-7 cells transfected with Kir2.1-His6, Kir2.4-FLAG or both The constructs transfected and antibody probes used are indicated at the bottom. + indicates construct transfected, – indicates construct not present. Primary antibodies are designated by F for anti-FLAG, 2.1 for anti-Kir2.1 and H for anti-His. Arrows on the left indicate molecular masses of specific bands detected by specific primary antibodies. Molecular masses are given in kilodaltons (kDa). Lane 1 is a Western blot of Cos-7 cell lysate transfected with Kir2.4-FLAG and purified on a FLAG-Affinity gel (Sigma Chemical Co.). Cell lysates in lanes 2 to 7 were subjected to His 6 -pulldown prior to incubation with the primary antibody. Lanes 2 and 3 were obtained with a lysate of cells transfected with Kir2.1-His 6 only and lane 4 was obtained with a lysate of cells co-transfected with Kir2.4-FLAG and Kir2.1-His 6 . Lane 5 was obtained from cells transfected with Kir2.1-His 6 and incubated with anti-FLAG after His-pulldown. Lane 6 is a Western blot of lysate of Cos-7 cells transfected with Kir2.4-FLAG only incubated with anti-FLAG. Lane 7 is a Western blot with crude (non-transfected) Cos-7 cell lysate incubated with anti-Kir2.1.

    Techniques Used: Western Blot, Transfection, Construct, Purification, Incubation

    14) Product Images from "Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes"

    Article Title: Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes

    Journal: Channels

    doi: 10.1080/19336950.2016.1228498

    Kir2.1 protein exparession and localization in wt and dystrophic ventricular cardiomyocytes. (A) Representative western blot experiment of membrane lysates from adult wt and dystrophic (mdx and mdx-utr) ventricular tissues stained for Kir2.1 and the β-subunit of a G s protein (AS7). The latter was used as loading control. (B) Relative band intensities of Kir2.1 normalized to the respective band intensities of the loading control plotted as means ± SEM for wt (n = 5) and dystrophic (mdx, n = 6; mdx-utr, n = 2) animals. A Student's t-test did not reveal a significant difference between wt and mdx (p = 0.11; n.s., not significant). (C) Typical examples of Kir2.1 immunostainings of an isolated wt (left) and mdx (right) cardiomyocyte.
    Figure Legend Snippet: Kir2.1 protein exparession and localization in wt and dystrophic ventricular cardiomyocytes. (A) Representative western blot experiment of membrane lysates from adult wt and dystrophic (mdx and mdx-utr) ventricular tissues stained for Kir2.1 and the β-subunit of a G s protein (AS7). The latter was used as loading control. (B) Relative band intensities of Kir2.1 normalized to the respective band intensities of the loading control plotted as means ± SEM for wt (n = 5) and dystrophic (mdx, n = 6; mdx-utr, n = 2) animals. A Student's t-test did not reveal a significant difference between wt and mdx (p = 0.11; n.s., not significant). (C) Typical examples of Kir2.1 immunostainings of an isolated wt (left) and mdx (right) cardiomyocyte.

    Techniques Used: Western Blot, Staining, Isolation

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    Alomone Labs anti kir2 1
    Alterations of protein and mRNA levels of I K1 and I to revealed by Western blot analysis and real-time RT-PCR. (A) Top, examples of Western blot bands of <t>Kir2.1;</t> bottom, quantification of Kir2.1 (B) Top, examples of Western blot bands of Kv4.2; bottom,
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    Alterations of protein and mRNA levels of I K1 and I to revealed by Western blot analysis and real-time RT-PCR. (A) Top, examples of Western blot bands of Kir2.1; bottom, quantification of Kir2.1 (B) Top, examples of Western blot bands of Kv4.2; bottom,

    Journal: Molecular Medicine

    Article Title: Overexpression of M3 Muscarinic Receptor Is a Novel Strategy for Preventing Sudden Cardiac Death in Transgenic Mice

    doi: 10.2119/molmed.2011.00093

    Figure Lengend Snippet: Alterations of protein and mRNA levels of I K1 and I to revealed by Western blot analysis and real-time RT-PCR. (A) Top, examples of Western blot bands of Kir2.1; bottom, quantification of Kir2.1 (B) Top, examples of Western blot bands of Kv4.2; bottom,

    Article Snippet: The primary antibodies of anti-Kir2.1, anti-Kv4.2 (Alomone Labs, Jerusalme, Israel), and anti-Cav1.2, anti-M3 (Santa Cruz Technology, Santa Cruz, CA, USA), were used, with GAPDH (anti-GAPDH antibody from Kangcheng, Shanghai, China) as an internal control.

    Techniques: Western Blot, Quantitative RT-PCR

    Localization of inward rectifier potassium channel Kir2.1 in mouse retina. (A) Immunostaining for Kir2.1 (rabbit antibody; red) with DAPI counterstaining (blue) and PSD95 (green) to label photoreceptor terminals. Labels for retinal nuclear layers are as in Figure 1 . Kir2.1 is diffusely present in the IPL, but strongly labeled at the inner limiting membrane (arrowheads) and in the OPL. Five μm confocal stack. (B) Higher magnification view of the OPL reveals Kir2.1 (red) labeling largely below the photoreceptor terminals (green; PSD95 labeling), but with some diffuse and punctate labeling among the terminals. Two μm confocal stack. (Ci) Kir2.1 labeling (guinea pig antibody; red) colocalizes with Calbindin labeled horizontal cells (green) in the OPL. (Cii) Kir2.1 labeling in isolation. (Di) Another view of horizontal cells in the OPL (Calbindin labeling; green) along with mGluR6 labeling (magenta) to show locations of On bipolar cell dendritic tips. Clusters of mGluR6 label indicate cone terminals; three examples are indicated with paired arrowheads. Six μm confocal stack. (Dii) Kir2.1 labeling (rabbit antibody; red) in the same section. (Diii) Merged view of all three labels. Kir2.1 shows both diffuse and punctate labeling in the vicinity of horizontal cell axon terminal projections contacting rods, but little labeling near clusters of dendritic processes contacting cones. (Ei) Higher magnification view of a horizontal cell labeled with Calbindin antibody (green). Several representative axon terminal tips are highlighted with arrowheads. One μm confocal stack. (Eii) Kir2.1 labeling in the same section. (Eiii) Merged view of the two labels shows that tips of horizontal cell axon terminal processes contain punctate clusters of Kir2.1 labeling (arrowheads). (F) Average mRNA expression levels of Kir2.1 (Kcnj2) and kainate receptor subunits GluR6 (Grik2) and GluR7 (Grik3) in mouse retina from single-cell transcriptome data. Kir2.1 is most prominently expressed in horizontal cells and essentially absent from Müller glia. The kainate receptor subunit GluR6 is also found in horizontal cells and Off bipolar cells, but GluR7 is absent from these cell types. Data adapted from Hoang et al. (2020) .

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex

    doi: 10.3389/fncel.2021.667046

    Figure Lengend Snippet: Localization of inward rectifier potassium channel Kir2.1 in mouse retina. (A) Immunostaining for Kir2.1 (rabbit antibody; red) with DAPI counterstaining (blue) and PSD95 (green) to label photoreceptor terminals. Labels for retinal nuclear layers are as in Figure 1 . Kir2.1 is diffusely present in the IPL, but strongly labeled at the inner limiting membrane (arrowheads) and in the OPL. Five μm confocal stack. (B) Higher magnification view of the OPL reveals Kir2.1 (red) labeling largely below the photoreceptor terminals (green; PSD95 labeling), but with some diffuse and punctate labeling among the terminals. Two μm confocal stack. (Ci) Kir2.1 labeling (guinea pig antibody; red) colocalizes with Calbindin labeled horizontal cells (green) in the OPL. (Cii) Kir2.1 labeling in isolation. (Di) Another view of horizontal cells in the OPL (Calbindin labeling; green) along with mGluR6 labeling (magenta) to show locations of On bipolar cell dendritic tips. Clusters of mGluR6 label indicate cone terminals; three examples are indicated with paired arrowheads. Six μm confocal stack. (Dii) Kir2.1 labeling (rabbit antibody; red) in the same section. (Diii) Merged view of all three labels. Kir2.1 shows both diffuse and punctate labeling in the vicinity of horizontal cell axon terminal projections contacting rods, but little labeling near clusters of dendritic processes contacting cones. (Ei) Higher magnification view of a horizontal cell labeled with Calbindin antibody (green). Several representative axon terminal tips are highlighted with arrowheads. One μm confocal stack. (Eii) Kir2.1 labeling in the same section. (Eiii) Merged view of the two labels shows that tips of horizontal cell axon terminal processes contain punctate clusters of Kir2.1 labeling (arrowheads). (F) Average mRNA expression levels of Kir2.1 (Kcnj2) and kainate receptor subunits GluR6 (Grik2) and GluR7 (Grik3) in mouse retina from single-cell transcriptome data. Kir2.1 is most prominently expressed in horizontal cells and essentially absent from Müller glia. The kainate receptor subunit GluR6 is also found in horizontal cells and Off bipolar cells, but GluR7 is absent from these cell types. Data adapted from Hoang et al. (2020) .

    Article Snippet: Antibodies used included rabbit anti-SAP-102 (Thermo Fisher Scientific, Waltham, MA; AB_2546592 ), goat anti-SAP-102 (Abcam, Cambridge, MA; AB_777828 ), mouse anti-SAP-97 clone K64/15 (UC Davis/NIH NeuroMab Facility, Davis, CA; AB_2091920 ), mouse IgG1 anti-Chapsyn-110 clone N18/30 (UC Davis/NIH NeuroMab Facility; AB_2277296 ), mouse IgG2a anti-PSD-95 clone K28/43 (UC Davis/NIH NeuroMab Facility; AB_444362 ), mouse anti-CASK (Antibodies-online, Inc., Limerick, PA), sheep anti-mGluR6 (gift of Dr. Catherine Morgans; ), mouse anti-GluR6/7 clone NL9 (Millipore-Sigma, Burlington, MA; Cat# 04-921, AB_1587072 ), rabbit anti-Kir2.1 (Alomone, Jerusalem, Israel; AB_2040107 ), guinea pig anti-Kir2.1 (Alomone; AB_2340970 ), rabbit anti-Connexin 57 (Invitrogen, Camarillo, CA; AB_2314266 ), rabbit anti-Pannexin 1 (Alomone; AB_2340917 ), rabbit anti-Pannexin 2 (Thermo Fisher Scioentific; AB_2533518 ), rabbit anti-Calbindin (Swant, Bellinzona, Switzerland; AB_1000034 ), mouse anti-Calbindin (Abcam; AB_1658451 ), mouse anti-Glutamine Synthetase (Millipore-Sigma; AB_2110656 ), and rabbit anti-SLC18B1 (Sigma Life Sciences, St. Louis, MO; AB_10600797 ).

    Techniques: Immunostaining, Labeling, Isolation, Expressing

    Direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3. (A) Direct interaction between miR‐195 and Cavβ1. A fragment of miR‐195 that binds to CACNB1. miR‐195 is complementary to the CACNB1 gene 943‐949, which encodes Cavβ1, and the corresponding mutant sequence is designed based on the binding site. (B) Luciferase reporter with a CACNB1 fragment capable of binding to miR‐195. The gene was co‐transfected with miR‐195 into HEK293 cells, and miR‐195 reduced the activity of the luciferase reporter gene, ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels, et al. Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels

    doi: 10.1111/jcmm.15431

    Figure Lengend Snippet: Direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3. (A) Direct interaction between miR‐195 and Cavβ1. A fragment of miR‐195 that binds to CACNB1. miR‐195 is complementary to the CACNB1 gene 943‐949, which encodes Cavβ1, and the corresponding mutant sequence is designed based on the binding site. (B) Luciferase reporter with a CACNB1 fragment capable of binding to miR‐195. The gene was co‐transfected with miR‐195 into HEK293 cells, and miR‐195 reduced the activity of the luciferase reporter gene, ** P

    Article Snippet: 2.12 ImmunocytochemistryCultured NMVCs were incubated with anti‐Cavβ1 (Cat# ab85020;Abcam, Cambridge, UK) and antibody of Kir2.1 (Alomone Labs, Cat# APC‐026), antibody of Kv4.3 (Alomone Labs, Cat#APC‐017), antibody of α‐actinin (sigma, Cat#A7811) at 4°C refrigerator for overnight.

    Techniques: Mutagenesis, Sequencing, Binding Assay, Luciferase, Transfection, Activity Assay

    miR‐195 inhibits the expression of Cavβ1, Kir2.1 and Kv4.3 in cardiomyocytes by immunofluorescence and Western blot. (A) Effects of miR‐195 on protein levels of endogenous Cavβ1 in primary cultured cardiomyocytes by Western blot analysis. miR‐195 effectively inhibited the expression of Cavβ1 relative to control group, whereas the scrambled NC miRNA failed to affect the protein levels. In contrast, AMO‐195 rescued the down‐regulation of Cavβ1 elicited by miR‐195. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels, et al. Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels

    doi: 10.1111/jcmm.15431

    Figure Lengend Snippet: miR‐195 inhibits the expression of Cavβ1, Kir2.1 and Kv4.3 in cardiomyocytes by immunofluorescence and Western blot. (A) Effects of miR‐195 on protein levels of endogenous Cavβ1 in primary cultured cardiomyocytes by Western blot analysis. miR‐195 effectively inhibited the expression of Cavβ1 relative to control group, whereas the scrambled NC miRNA failed to affect the protein levels. In contrast, AMO‐195 rescued the down‐regulation of Cavβ1 elicited by miR‐195. * P

    Article Snippet: 2.12 ImmunocytochemistryCultured NMVCs were incubated with anti‐Cavβ1 (Cat# ab85020;Abcam, Cambridge, UK) and antibody of Kir2.1 (Alomone Labs, Cat# APC‐026), antibody of Kv4.3 (Alomone Labs, Cat#APC‐017), antibody of α‐actinin (sigma, Cat#A7811) at 4°C refrigerator for overnight.

    Techniques: Expressing, Immunofluorescence, Western Blot, Cell Culture

    miR‐195 inhibits the protein expression of Cavβ1, Kir2.1 and Kv4.3 in vivo. (A‐C) qPCR showed the changes of CACNB1/KCNJ2/KCND3 transcripts in cardiac tissues from overexpressing miR‐195 mice, n = 5‐9. (D) Verification of the specificity of miR‐195 on Cavβ1. Compared with NC, the expression of Cavβ1 protein in the overexpressed miR‐195 group was decreased. ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels, et al. Up‐regulation of miR‐195 contributes to cardiac hypertrophy‐induced arrhythmia by targeting calcium and potassium channels

    doi: 10.1111/jcmm.15431

    Figure Lengend Snippet: miR‐195 inhibits the protein expression of Cavβ1, Kir2.1 and Kv4.3 in vivo. (A‐C) qPCR showed the changes of CACNB1/KCNJ2/KCND3 transcripts in cardiac tissues from overexpressing miR‐195 mice, n = 5‐9. (D) Verification of the specificity of miR‐195 on Cavβ1. Compared with NC, the expression of Cavβ1 protein in the overexpressed miR‐195 group was decreased. ** P

    Article Snippet: 2.12 ImmunocytochemistryCultured NMVCs were incubated with anti‐Cavβ1 (Cat# ab85020;Abcam, Cambridge, UK) and antibody of Kir2.1 (Alomone Labs, Cat# APC‐026), antibody of Kv4.3 (Alomone Labs, Cat#APC‐017), antibody of α‐actinin (sigma, Cat#A7811) at 4°C refrigerator for overnight.

    Techniques: Expressing, In Vivo, Real-time Polymerase Chain Reaction, Mouse Assay

    Ion channels in cardiac MΦ. Current inhibition in the presence of 1 and 10 nM MgTx ( n =15, N =6) ( A and C ) or 3 µM XEN-D0103 ( n =8, N =6) ( B and D ). ( E ) Cumulative inactivation of Type 1 outward currents was abolished after MgTx application. ( F ) Lack of cumulative inactivation was observed in MΦ with Type 2 outward current profile. ( G ) Representative recordings of steady-state inactivation for Types 1 and 2 outward current profiles. ( H ) Steady-state activation and inactivation curves for Type 1 outward currents plotted as a function of holding potential. ( I ) Flow cytometry quantification of cell surface expression of Kv1.3 (in 86.0±3.0% of FACS-purified cardiac MΦ), Kv1.5 (84.2±1.8%) and Kir2.1 (55.9±11.8%) ( N =5); negative control data are from same cells and labels, omitting the primary antibody (

    Journal: Cardiovascular Research

    Article Title: Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels

    doi: 10.1093/cvr/cvab126

    Figure Lengend Snippet: Ion channels in cardiac MΦ. Current inhibition in the presence of 1 and 10 nM MgTx ( n =15, N =6) ( A and C ) or 3 µM XEN-D0103 ( n =8, N =6) ( B and D ). ( E ) Cumulative inactivation of Type 1 outward currents was abolished after MgTx application. ( F ) Lack of cumulative inactivation was observed in MΦ with Type 2 outward current profile. ( G ) Representative recordings of steady-state inactivation for Types 1 and 2 outward current profiles. ( H ) Steady-state activation and inactivation curves for Type 1 outward currents plotted as a function of holding potential. ( I ) Flow cytometry quantification of cell surface expression of Kv1.3 (in 86.0±3.0% of FACS-purified cardiac MΦ), Kv1.5 (84.2±1.8%) and Kir2.1 (55.9±11.8%) ( N =5); negative control data are from same cells and labels, omitting the primary antibody (

    Article Snippet: Rabbit-anti-Kv 1.3, anti-Kv 1.5, anti-Kir2.1 primary antibodies (1:500; all from Alomone Labs), or rabbit-anti-Cx43 antibody (1:400, Abcam ab11370) were added to the cell suspension and incubated for 30 min at 4°C.

    Techniques: Inhibition, Activation Assay, Flow Cytometry, Expressing, FACS, Purification, Negative Control

    Deconstruction of simulated currents. ( A ) Cardiac MΦ with Type 1 or 2 outward current profiles in the presence of an inward-rectifier current can be reproduced by a background current ( I b ), an outward current ( I outward, I Kv1.3 for Type 1, left, or I Kv1.5 for Type 2, right) and an inward-rectifier current ( I Kir2.1 ). Comparison between simulated (red lines) and experimentally recorded currents (black traces). ( B ) Deconstruction of the different simulated currents. ( C ) Simulated IV curve after adding the three currents represented in ( B ).

    Journal: Cardiovascular Research

    Article Title: Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels

    doi: 10.1093/cvr/cvab126

    Figure Lengend Snippet: Deconstruction of simulated currents. ( A ) Cardiac MΦ with Type 1 or 2 outward current profiles in the presence of an inward-rectifier current can be reproduced by a background current ( I b ), an outward current ( I outward, I Kv1.3 for Type 1, left, or I Kv1.5 for Type 2, right) and an inward-rectifier current ( I Kir2.1 ). Comparison between simulated (red lines) and experimentally recorded currents (black traces). ( B ) Deconstruction of the different simulated currents. ( C ) Simulated IV curve after adding the three currents represented in ( B ).

    Article Snippet: Rabbit-anti-Kv 1.3, anti-Kv 1.5, anti-Kir2.1 primary antibodies (1:500; all from Alomone Labs), or rabbit-anti-Cx43 antibody (1:400, Abcam ab11370) were added to the cell suspension and incubated for 30 min at 4°C.

    Techniques: