kir2 1  (Alomone Labs)


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    Structured Review

    Alomone Labs kir2 1
    <t>Kir2.1</t> WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Kir2 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kir2 1 - by Bioz Stars, 2022-01
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    Images

    1) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    2) Product Images from "Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype"

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddu201

    Mutation detection by sequence analysis of the KCNJ2 coding region. ( A ) Pedigree of the family harboring a novel mutations in KCNJ2 . Squares are males and circles females; solid black symbols represent propositi and slash deceased individual. ( B ) ECG recording from propositi showing shortened QT interval (heart rate, 87 bpm; QT 275 ms; paper speed 25 mm/s). ( C ) Electropherograms showing the heterozygous c.1037A > C transition (arrow-headed), predicting a novel non-synonymous p.K346T variant in propositi compared with the sequence of a healthy individual (WT). ( D ) Alignments of several KCNJ2 sequences flanking the K346T substitution (K/T, arrow-headed) showing that this residue is highly conserved in several vertebrate species. ( E ) Schematic representation of the membrane topology of a human Kir2.1 subunit indicating the position of the p.K346T variant.
    Figure Legend Snippet: Mutation detection by sequence analysis of the KCNJ2 coding region. ( A ) Pedigree of the family harboring a novel mutations in KCNJ2 . Squares are males and circles females; solid black symbols represent propositi and slash deceased individual. ( B ) ECG recording from propositi showing shortened QT interval (heart rate, 87 bpm; QT 275 ms; paper speed 25 mm/s). ( C ) Electropherograms showing the heterozygous c.1037A > C transition (arrow-headed), predicting a novel non-synonymous p.K346T variant in propositi compared with the sequence of a healthy individual (WT). ( D ) Alignments of several KCNJ2 sequences flanking the K346T substitution (K/T, arrow-headed) showing that this residue is highly conserved in several vertebrate species. ( E ) Schematic representation of the membrane topology of a human Kir2.1 subunit indicating the position of the p.K346T variant.

    Techniques Used: Mutagenesis, Sequencing, Variant Assay

    Characterization of astrocytoma cells expressing WT and K346T channels. Co-immunofluorescences of cells expressing WT ( A ) or K346T ( B ) channels with anti-Kir2.1 pAb (red) and FITC-conjugated phallacidin (green) show that WT channels are localized in perinuclear vesicles (short arrows in A) and occasionally at plasma membranes (long arrows in A), while mutated channels are mainly expressed at plasma membranes (long arrows in B). Scale bar: 10 μm. ( C ) RT-PCR analysis of Kir2.1 mRNA in WT (1), K346T (2) channel or empty-vector expressing U251 cell lines (3). GAPDH housekeeping gene normalizes the amount of template. ( D ) WB analysis of membrane (MEM) and cytosolic (CYT) proteins derived from WT or K346T Kir2.1-expressing cells after Histidine co-purification. Molecular weight markers are on the left (kDa). ( E ) Densitometric analysis of protein bands from four independent experiments (mean ± SEM, * P
    Figure Legend Snippet: Characterization of astrocytoma cells expressing WT and K346T channels. Co-immunofluorescences of cells expressing WT ( A ) or K346T ( B ) channels with anti-Kir2.1 pAb (red) and FITC-conjugated phallacidin (green) show that WT channels are localized in perinuclear vesicles (short arrows in A) and occasionally at plasma membranes (long arrows in A), while mutated channels are mainly expressed at plasma membranes (long arrows in B). Scale bar: 10 μm. ( C ) RT-PCR analysis of Kir2.1 mRNA in WT (1), K346T (2) channel or empty-vector expressing U251 cell lines (3). GAPDH housekeeping gene normalizes the amount of template. ( D ) WB analysis of membrane (MEM) and cytosolic (CYT) proteins derived from WT or K346T Kir2.1-expressing cells after Histidine co-purification. Molecular weight markers are on the left (kDa). ( E ) Densitometric analysis of protein bands from four independent experiments (mean ± SEM, * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Derivative Assay, Copurification, Molecular Weight

    The K346T mutation affects the distribution of Kir2.1 channels in membrane lipid rafts. ( A ) WB analysis of cholesterol-rich (triton insoluble fractions: 3–5) and cholesterol-poor membrane fractions (triton soluble fractions: 10–12) of WT or K346T Kir2.1-expressing cells. WT channels are mainly distributed in triton insoluble fractions (gray box), whereas K346T is also abundantly localized in cholesterol-poor fractions (black boxes). Cav-1 and flotillin-1 identify the caveolar raft fractions. Molecular weight markers are on the left (kDa). ( B–E ) Normal distributions of total protein (indicated on top) in membrane fractions isolated by sucrose density gradient. The levels of protein in each fraction are normalized to the total protein amount recovered from all the fractions together.
    Figure Legend Snippet: The K346T mutation affects the distribution of Kir2.1 channels in membrane lipid rafts. ( A ) WB analysis of cholesterol-rich (triton insoluble fractions: 3–5) and cholesterol-poor membrane fractions (triton soluble fractions: 10–12) of WT or K346T Kir2.1-expressing cells. WT channels are mainly distributed in triton insoluble fractions (gray box), whereas K346T is also abundantly localized in cholesterol-poor fractions (black boxes). Cav-1 and flotillin-1 identify the caveolar raft fractions. Molecular weight markers are on the left (kDa). ( B–E ) Normal distributions of total protein (indicated on top) in membrane fractions isolated by sucrose density gradient. The levels of protein in each fraction are normalized to the total protein amount recovered from all the fractions together.

    Techniques Used: Mutagenesis, Western Blot, Expressing, Molecular Weight, Isolation

    The K346T mutation increases Kir2.1 currents. Sample current families recorded from oocytes expressing WT ( A ) or K346T ( B ) mRNA (the pulse protocol is shown as inset). ( C ) I–V relationships for WT (white circles, 3 ng) and K346T (black circles, 3 ng) channels. ( D ) Current amplitudes recorded at −100 mV from cells injected with the indicated mRNAs whose amounts are reported in brackets (mean ± SEM; n = 120; * P
    Figure Legend Snippet: The K346T mutation increases Kir2.1 currents. Sample current families recorded from oocytes expressing WT ( A ) or K346T ( B ) mRNA (the pulse protocol is shown as inset). ( C ) I–V relationships for WT (white circles, 3 ng) and K346T (black circles, 3 ng) channels. ( D ) Current amplitudes recorded at −100 mV from cells injected with the indicated mRNAs whose amounts are reported in brackets (mean ± SEM; n = 120; * P

    Techniques Used: Mutagenesis, Expressing, Injection

    The K346T mutation increases protein stability. ( A ) WB analysis of protein extracts derived from cells expressing WT and K346T channels treated with the protein synthesis inhibitor cycloheximide for 3, 6 and 12 h. WT protein degradation is almost complete after 12 h treatment, while K346T protein is still detectable at this time. Actin is used as loading control. Molecular weight markers are on the left (kDa). ( B ) Densitometric analysis of protein bands normalized with respect to the amount of either WT (white bar) or K346T (gray bar) Kir2.1 protein in control conditions. Data are expressed as mean ± SEM from four independent experiments (*** P
    Figure Legend Snippet: The K346T mutation increases protein stability. ( A ) WB analysis of protein extracts derived from cells expressing WT and K346T channels treated with the protein synthesis inhibitor cycloheximide for 3, 6 and 12 h. WT protein degradation is almost complete after 12 h treatment, while K346T protein is still detectable at this time. Actin is used as loading control. Molecular weight markers are on the left (kDa). ( B ) Densitometric analysis of protein bands normalized with respect to the amount of either WT (white bar) or K346T (gray bar) Kir2.1 protein in control conditions. Data are expressed as mean ± SEM from four independent experiments (*** P

    Techniques Used: Mutagenesis, Western Blot, Derivative Assay, Expressing, Molecular Weight

    The K346T mutation reduces Kir2.1 channels interaction with Cav-2. ( A ) WB analysis of Kir2.1 channel's interactors after Histidine (His) co-purification of astrocytoma cells expressing WT or K346T channels. Input lanes represent protein extracts before His co-purification. WT and K346T channels co-purify similarly with Cav-1, whereas the K346T mutation reduces the association with Cav-2. One representative experiment out of three is shown. Molecular weight markers are indicated on the left (kDa). ( B ) Densitometric analysis (ratio) of protein bands corresponding to Cav-1 and Cav-2 normalized respect to WT (white bars) or K346T (gray bars) Kir2.1 protein levels. Data are expressed as mean ± SEM from three independent experiments (*** P
    Figure Legend Snippet: The K346T mutation reduces Kir2.1 channels interaction with Cav-2. ( A ) WB analysis of Kir2.1 channel's interactors after Histidine (His) co-purification of astrocytoma cells expressing WT or K346T channels. Input lanes represent protein extracts before His co-purification. WT and K346T channels co-purify similarly with Cav-1, whereas the K346T mutation reduces the association with Cav-2. One representative experiment out of three is shown. Molecular weight markers are indicated on the left (kDa). ( B ) Densitometric analysis (ratio) of protein bands corresponding to Cav-1 and Cav-2 normalized respect to WT (white bars) or K346T (gray bars) Kir2.1 protein levels. Data are expressed as mean ± SEM from three independent experiments (*** P

    Techniques Used: Mutagenesis, Western Blot, Copurification, Expressing, Molecular Weight

    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 "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

    5) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    13) 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

    14) 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

    15) 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

    16) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    17) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    18) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    19) 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

    20) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    21) 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

    22) 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

    23) 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

    24) 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

    25) Product Images from "Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents"

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA120.002071

    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Figure Legend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Techniques Used: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P
    Figure Legend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Techniques Used: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.
    Figure Legend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Techniques Used: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).
    Figure Legend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Techniques Used:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.
    Figure Legend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Techniques Used: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

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    Alomone Labs kir2 1
    <t>Kir2.1</t> WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.
    Kir2 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    doi: 10.1074/mcp.RA120.002071

    Figure Lengend Snippet: Kir2.1 WT versus Kir2.1 Δ314-315 interactome profiling. Variations in the Kir2.1 WT and Kir2.1 Δ314-315 interactome profiles are visualized in a scatter plot representing the average Log 2 -transformed, normalized SpC counts for both the Kir2.1 WT ( y axis) and Kir2.1 Δ314-315 ( x axis) bait proteins. The Kir2.1 bait, Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as blue, green, red and gray dots, respectively.

    Article Snippet: This observation indicates that Kir2.1 not only interacts with other channels-transporters at the cytoplasmic membrane but that the co-trafficking of these proteins in the endoplasmic reticulum (ER) and Golgi apparatus is an important aspect of their function.

    Techniques: Transformation Assay

    PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    doi: 10.1074/mcp.RA120.002071

    Figure Lengend Snippet: PKP4 is a positive regulator of I Kir2.1 . Patch-clamping analyses in HEK293 cells upon genetic perturbation of PKP4 , i.e. ( A ) upon overexpression of PKP4 and ( B ) upon CRISPR/Cas9-mediated depletion of PKP4. * P

    Article Snippet: This observation indicates that Kir2.1 not only interacts with other channels-transporters at the cytoplasmic membrane but that the co-trafficking of these proteins in the endoplasmic reticulum (ER) and Golgi apparatus is an important aspect of their function.

    Techniques: Over Expression, CRISPR

    Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    doi: 10.1074/mcp.RA120.002071

    Figure Lengend Snippet: Kir2.1 and PKP4 co-localize in adult ventricular myocytes. Immunofluorescence (IF) staining analyses of the subcellular localization of Kir2.1 ( A , red ), Actinin ( B , green ) and PKP4 ( C , light blue ) in a freshly isolated rat adult ventricular myocytes. ( D ) Merge image. ( E ) Pixel intensity profile of PKP4, Kir2.1 and actinin along a line in the merge image, i.e. blue arrow shown in ( D ) showing the striated co-localization of the three proteins at the z-disks near the cardiac sarcomeres. ( F ) Differential interference contrast (DIC) image of the myocyte. ( G – J ) Zoomed in images of the intercalated disks. Scale bars: 10 μ m . ID: intercalated disk.

    Article Snippet: This observation indicates that Kir2.1 not only interacts with other channels-transporters at the cytoplasmic membrane but that the co-trafficking of these proteins in the endoplasmic reticulum (ER) and Golgi apparatus is an important aspect of their function.

    Techniques: Immunofluorescence, Staining, Isolation

    Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    doi: 10.1074/mcp.RA120.002071

    Figure Lengend Snippet: Graphical representation of the Kir2.1 BioID interactome. Protein complexes and groups of functionally related proteins encompassing 152 out of the 218 high-confidence Kir2.1 BioID hits are depicted in a cell. Major organelles in the cell (nucleus, endoplasmic reticulum, Golgi apparatus and cytoplasmic membrane) are shown (light gray) in the background to roughly indicate the approximate subcellular localization of the proteins in the cell. The Kir2.1 WT -preferred interactors, Kir2.1 Δ314-315 -preferred interactors and Kir2.1 WT/Δ314-315 -neutral interactors are represented as green, red and gray circles, respectively. The color intensity of each circle is an indicator of the strength of the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ” value. The size of each circle represents the average SpC counts observed in either the Kir2.1 WT or Kir2.1 Δ314-315 BioID experiment (whichever is the largest is represented in the figure).

    Article Snippet: This observation indicates that Kir2.1 not only interacts with other channels-transporters at the cytoplasmic membrane but that the co-trafficking of these proteins in the endoplasmic reticulum (ER) and Golgi apparatus is an important aspect of their function.

    Techniques:

    Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

    doi: 10.1074/mcp.RA120.002071

    Figure Lengend Snippet: Overall procedure to generate the Kir2.1 BioID interactome map. Stable cells expressing BirA*-tagged Kir2.1 WT , Kir2.1 Δ314-315 or TM-CTRL bait proteins were generated using the Flp-In T-Rex 293 cell line. Expression of the bait proteins was induced by tetracycline and cells were treated with Supplemental biotin for 24 h. After cell lysis, biotinylated proteins were purified on streptavidin-agarose beads and digested with trypsin. Tryptic peptides were analyzed using LC–MS/MS and proteins were identified using Proteome Discoverer. After applying a stringent set of criteria, we identified 218 high-confidence Kir2.1 BioID hits. Using the normalized Kir2.1 WT/Δ314-315 SpC ratio “ R ”, we classified the interactors in three categories: 75 Kir2.1 WT -preferred interactors, 66 Kir2.1 Δ314-315 -preferred interactors and 77 Kir2.1 WT/Δ314-315 -neutral interactors. CTRL: control; LC–MS/MS: liquid chromatography with tandem MS; SAINT: Significance Analysis of INTeractome; SpC: spectral counts; TM: transmembrane; WT: WT.

    Article Snippet: This observation indicates that Kir2.1 not only interacts with other channels-transporters at the cytoplasmic membrane but that the co-trafficking of these proteins in the endoplasmic reticulum (ER) and Golgi apparatus is an important aspect of their function.

    Techniques: Expressing, Generated, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography

    Mutation detection by sequence analysis of the KCNJ2 coding region. ( A ) Pedigree of the family harboring a novel mutations in KCNJ2 . Squares are males and circles females; solid black symbols represent propositi and slash deceased individual. ( B ) ECG recording from propositi showing shortened QT interval (heart rate, 87 bpm; QT 275 ms; paper speed 25 mm/s). ( C ) Electropherograms showing the heterozygous c.1037A > C transition (arrow-headed), predicting a novel non-synonymous p.K346T variant in propositi compared with the sequence of a healthy individual (WT). ( D ) Alignments of several KCNJ2 sequences flanking the K346T substitution (K/T, arrow-headed) showing that this residue is highly conserved in several vertebrate species. ( E ) Schematic representation of the membrane topology of a human Kir2.1 subunit indicating the position of the p.K346T variant.

    Journal: Human Molecular Genetics

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    doi: 10.1093/hmg/ddu201

    Figure Lengend Snippet: Mutation detection by sequence analysis of the KCNJ2 coding region. ( A ) Pedigree of the family harboring a novel mutations in KCNJ2 . Squares are males and circles females; solid black symbols represent propositi and slash deceased individual. ( B ) ECG recording from propositi showing shortened QT interval (heart rate, 87 bpm; QT 275 ms; paper speed 25 mm/s). ( C ) Electropherograms showing the heterozygous c.1037A > C transition (arrow-headed), predicting a novel non-synonymous p.K346T variant in propositi compared with the sequence of a healthy individual (WT). ( D ) Alignments of several KCNJ2 sequences flanking the K346T substitution (K/T, arrow-headed) showing that this residue is highly conserved in several vertebrate species. ( E ) Schematic representation of the membrane topology of a human Kir2.1 subunit indicating the position of the p.K346T variant.

    Article Snippet: Homology modeling and cholesterol docking The 3D structure of Kir2.1 was built by comparative modeling using the software Modeller® ( ).

    Techniques: Mutagenesis, Sequencing, Variant Assay

    Characterization of astrocytoma cells expressing WT and K346T channels. Co-immunofluorescences of cells expressing WT ( A ) or K346T ( B ) channels with anti-Kir2.1 pAb (red) and FITC-conjugated phallacidin (green) show that WT channels are localized in perinuclear vesicles (short arrows in A) and occasionally at plasma membranes (long arrows in A), while mutated channels are mainly expressed at plasma membranes (long arrows in B). Scale bar: 10 μm. ( C ) RT-PCR analysis of Kir2.1 mRNA in WT (1), K346T (2) channel or empty-vector expressing U251 cell lines (3). GAPDH housekeeping gene normalizes the amount of template. ( D ) WB analysis of membrane (MEM) and cytosolic (CYT) proteins derived from WT or K346T Kir2.1-expressing cells after Histidine co-purification. Molecular weight markers are on the left (kDa). ( E ) Densitometric analysis of protein bands from four independent experiments (mean ± SEM, * P

    Journal: Human Molecular Genetics

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    doi: 10.1093/hmg/ddu201

    Figure Lengend Snippet: Characterization of astrocytoma cells expressing WT and K346T channels. Co-immunofluorescences of cells expressing WT ( A ) or K346T ( B ) channels with anti-Kir2.1 pAb (red) and FITC-conjugated phallacidin (green) show that WT channels are localized in perinuclear vesicles (short arrows in A) and occasionally at plasma membranes (long arrows in A), while mutated channels are mainly expressed at plasma membranes (long arrows in B). Scale bar: 10 μm. ( C ) RT-PCR analysis of Kir2.1 mRNA in WT (1), K346T (2) channel or empty-vector expressing U251 cell lines (3). GAPDH housekeeping gene normalizes the amount of template. ( D ) WB analysis of membrane (MEM) and cytosolic (CYT) proteins derived from WT or K346T Kir2.1-expressing cells after Histidine co-purification. Molecular weight markers are on the left (kDa). ( E ) Densitometric analysis of protein bands from four independent experiments (mean ± SEM, * P

    Article Snippet: Homology modeling and cholesterol docking The 3D structure of Kir2.1 was built by comparative modeling using the software Modeller® ( ).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Derivative Assay, Copurification, Molecular Weight

    The K346T mutation affects the distribution of Kir2.1 channels in membrane lipid rafts. ( A ) WB analysis of cholesterol-rich (triton insoluble fractions: 3–5) and cholesterol-poor membrane fractions (triton soluble fractions: 10–12) of WT or K346T Kir2.1-expressing cells. WT channels are mainly distributed in triton insoluble fractions (gray box), whereas K346T is also abundantly localized in cholesterol-poor fractions (black boxes). Cav-1 and flotillin-1 identify the caveolar raft fractions. Molecular weight markers are on the left (kDa). ( B–E ) Normal distributions of total protein (indicated on top) in membrane fractions isolated by sucrose density gradient. The levels of protein in each fraction are normalized to the total protein amount recovered from all the fractions together.

    Journal: Human Molecular Genetics

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    doi: 10.1093/hmg/ddu201

    Figure Lengend Snippet: The K346T mutation affects the distribution of Kir2.1 channels in membrane lipid rafts. ( A ) WB analysis of cholesterol-rich (triton insoluble fractions: 3–5) and cholesterol-poor membrane fractions (triton soluble fractions: 10–12) of WT or K346T Kir2.1-expressing cells. WT channels are mainly distributed in triton insoluble fractions (gray box), whereas K346T is also abundantly localized in cholesterol-poor fractions (black boxes). Cav-1 and flotillin-1 identify the caveolar raft fractions. Molecular weight markers are on the left (kDa). ( B–E ) Normal distributions of total protein (indicated on top) in membrane fractions isolated by sucrose density gradient. The levels of protein in each fraction are normalized to the total protein amount recovered from all the fractions together.

    Article Snippet: Homology modeling and cholesterol docking The 3D structure of Kir2.1 was built by comparative modeling using the software Modeller® ( ).

    Techniques: Mutagenesis, Western Blot, Expressing, Molecular Weight, Isolation

    The K346T mutation increases Kir2.1 currents. Sample current families recorded from oocytes expressing WT ( A ) or K346T ( B ) mRNA (the pulse protocol is shown as inset). ( C ) I–V relationships for WT (white circles, 3 ng) and K346T (black circles, 3 ng) channels. ( D ) Current amplitudes recorded at −100 mV from cells injected with the indicated mRNAs whose amounts are reported in brackets (mean ± SEM; n = 120; * P

    Journal: Human Molecular Genetics

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    doi: 10.1093/hmg/ddu201

    Figure Lengend Snippet: The K346T mutation increases Kir2.1 currents. Sample current families recorded from oocytes expressing WT ( A ) or K346T ( B ) mRNA (the pulse protocol is shown as inset). ( C ) I–V relationships for WT (white circles, 3 ng) and K346T (black circles, 3 ng) channels. ( D ) Current amplitudes recorded at −100 mV from cells injected with the indicated mRNAs whose amounts are reported in brackets (mean ± SEM; n = 120; * P

    Article Snippet: Homology modeling and cholesterol docking The 3D structure of Kir2.1 was built by comparative modeling using the software Modeller® ( ).

    Techniques: Mutagenesis, Expressing, Injection

    The K346T mutation increases protein stability. ( A ) WB analysis of protein extracts derived from cells expressing WT and K346T channels treated with the protein synthesis inhibitor cycloheximide for 3, 6 and 12 h. WT protein degradation is almost complete after 12 h treatment, while K346T protein is still detectable at this time. Actin is used as loading control. Molecular weight markers are on the left (kDa). ( B ) Densitometric analysis of protein bands normalized with respect to the amount of either WT (white bar) or K346T (gray bar) Kir2.1 protein in control conditions. Data are expressed as mean ± SEM from four independent experiments (*** P

    Journal: Human Molecular Genetics

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    doi: 10.1093/hmg/ddu201

    Figure Lengend Snippet: The K346T mutation increases protein stability. ( A ) WB analysis of protein extracts derived from cells expressing WT and K346T channels treated with the protein synthesis inhibitor cycloheximide for 3, 6 and 12 h. WT protein degradation is almost complete after 12 h treatment, while K346T protein is still detectable at this time. Actin is used as loading control. Molecular weight markers are on the left (kDa). ( B ) Densitometric analysis of protein bands normalized with respect to the amount of either WT (white bar) or K346T (gray bar) Kir2.1 protein in control conditions. Data are expressed as mean ± SEM from four independent experiments (*** P

    Article Snippet: Homology modeling and cholesterol docking The 3D structure of Kir2.1 was built by comparative modeling using the software Modeller® ( ).

    Techniques: Mutagenesis, Western Blot, Derivative Assay, Expressing, Molecular Weight

    The K346T mutation reduces Kir2.1 channels interaction with Cav-2. ( A ) WB analysis of Kir2.1 channel's interactors after Histidine (His) co-purification of astrocytoma cells expressing WT or K346T channels. Input lanes represent protein extracts before His co-purification. WT and K346T channels co-purify similarly with Cav-1, whereas the K346T mutation reduces the association with Cav-2. One representative experiment out of three is shown. Molecular weight markers are indicated on the left (kDa). ( B ) Densitometric analysis (ratio) of protein bands corresponding to Cav-1 and Cav-2 normalized respect to WT (white bars) or K346T (gray bars) Kir2.1 protein levels. Data are expressed as mean ± SEM from three independent experiments (*** P

    Journal: Human Molecular Genetics

    Article Title: Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism–epilepsy phenotype

    doi: 10.1093/hmg/ddu201

    Figure Lengend Snippet: The K346T mutation reduces Kir2.1 channels interaction with Cav-2. ( A ) WB analysis of Kir2.1 channel's interactors after Histidine (His) co-purification of astrocytoma cells expressing WT or K346T channels. Input lanes represent protein extracts before His co-purification. WT and K346T channels co-purify similarly with Cav-1, whereas the K346T mutation reduces the association with Cav-2. One representative experiment out of three is shown. Molecular weight markers are indicated on the left (kDa). ( B ) Densitometric analysis (ratio) of protein bands corresponding to Cav-1 and Cav-2 normalized respect to WT (white bars) or K346T (gray bars) Kir2.1 protein levels. Data are expressed as mean ± SEM from three independent experiments (*** P

    Article Snippet: Homology modeling and cholesterol docking The 3D structure of Kir2.1 was built by comparative modeling using the software Modeller® ( ).

    Techniques: Mutagenesis, Western Blot, Copurification, Expressing, Molecular Weight

    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: The protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in the myocardium of HF mice compared with sham group. (2) Overexpression of miR‐195 by Lenti‐miR‐195 decreased the cardiac function in WT mice and increased the likelihood of arrhythmia induction and duration of arrhythmia in normal mice. (3) Lenti‐miR‐195 inhibitor can reverse the decreased cardiac function, the increased incidence of arrhythmia and prolonged duration of arrhythmia induced by TAC in mice. (4) The protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in mice after miR‐195 treatment. (5) Luciferase assay results showed direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3.

    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: The protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in the myocardium of HF mice compared with sham group. (2) Overexpression of miR‐195 by Lenti‐miR‐195 decreased the cardiac function in WT mice and increased the likelihood of arrhythmia induction and duration of arrhythmia in normal mice. (3) Lenti‐miR‐195 inhibitor can reverse the decreased cardiac function, the increased incidence of arrhythmia and prolonged duration of arrhythmia induced by TAC in mice. (4) The protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in mice after miR‐195 treatment. (5) Luciferase assay results showed direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3.

    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: The protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in the myocardium of HF mice compared with sham group. (2) Overexpression of miR‐195 by Lenti‐miR‐195 decreased the cardiac function in WT mice and increased the likelihood of arrhythmia induction and duration of arrhythmia in normal mice. (3) Lenti‐miR‐195 inhibitor can reverse the decreased cardiac function, the increased incidence of arrhythmia and prolonged duration of arrhythmia induced by TAC in mice. (4) The protein expressions of Cavβ1, Kir2.1 and Kv4.3 were decreased in mice after miR‐195 treatment. (5) Luciferase assay results showed direct interaction between miR‐195 and Cavβ1, Kir2.1 and Kv4.3.

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