kv11 1  (Alomone Labs)


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    Structured Review

    Alomone Labs kv11 1
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Kv11 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    kv11 1 - by Bioz Stars, 2024-06
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    Images

    1) Product Images from "Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation"

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018273

    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Figure Legend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing

    A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).
    Figure Legend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Techniques Used: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

    Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.
    Figure Legend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Techniques Used: Dominant Negative Mutation, Construct, Transfection, Activation Assay

    Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).
    Figure Legend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Techniques Used: Activation Assay

    The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.
    Figure Legend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Techniques Used: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

    Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.
    Figure Legend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence

    A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).
    Figure Legend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Techniques Used: Incubation, Western Blot, Cotransfection

    A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.
    Figure Legend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Techniques Used: Incubation, Expressing

    anti kv11 1 antibody  (Alomone Labs)


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    Alomone Labs anti kv11 1 antibody
    Anti Kv11 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    anti kv11 1 herg  (Alomone Labs)


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    Alomone Labs anti kv11 1 herg
    Anti Kv11 1 Herg, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    kv11 1 3 acc 003  (Alomone Labs)


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    Alomone Labs kv11 1 3 acc 003
    Kv11 1 3 Acc 003, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    kv11 1 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs kv11 1 antibody
    Kv11 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kv11 1 antibody/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    kv11 1  (Alomone Labs)


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    Alomone Labs kv11 1
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Kv11 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kv11 1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    kv11 1 - by Bioz Stars, 2024-06
    93/100 stars

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    1) Product Images from "Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation"

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018273

    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Figure Legend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing

    A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).
    Figure Legend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Techniques Used: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

    Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.
    Figure Legend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Techniques Used: Dominant Negative Mutation, Construct, Transfection, Activation Assay

    Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).
    Figure Legend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Techniques Used: Activation Assay

    The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.
    Figure Legend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Techniques Used: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

    Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.
    Figure Legend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence

    A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).
    Figure Legend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Techniques Used: Incubation, Western Blot, Cotransfection

    A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.
    Figure Legend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Techniques Used: Incubation, Expressing

    anti kv11 1 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti kv11 1 antibody
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Anti Kv11 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation"

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018273

    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Figure Legend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing

    A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).
    Figure Legend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Techniques Used: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

    Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.
    Figure Legend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Techniques Used: Dominant Negative Mutation, Construct, Transfection, Activation Assay

    Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).
    Figure Legend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Techniques Used: Activation Assay

    The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.
    Figure Legend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Techniques Used: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

    Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.
    Figure Legend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence

    A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).
    Figure Legend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Techniques Used: Incubation, Western Blot, Cotransfection

    A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.
    Figure Legend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Techniques Used: Incubation, Expressing

    surface membrane kv11 1  (Alomone Labs)


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    Alomone Labs surface membrane kv11 1
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Surface Membrane Kv11 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation"

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018273

    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
    Figure Legend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Techniques Used: Mutagenesis, Binding Assay, Sequencing

    A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).
    Figure Legend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Techniques Used: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

    Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.
    Figure Legend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Techniques Used: Dominant Negative Mutation, Construct, Transfection, Activation Assay

    Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).
    Figure Legend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Techniques Used: Activation Assay

    The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.
    Figure Legend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Techniques Used: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

    Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.
    Figure Legend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Techniques Used: Expressing, Transfection, Staining, Fluorescence

    A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).
    Figure Legend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Techniques Used: Incubation, Western Blot, Cotransfection

    A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.
    Figure Legend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Techniques Used: Incubation, Expressing

    anti kv11 1 herg extracellular fitc antibody  (Alomone Labs)


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    Alomone Labs anti kv11 1 herg extracellular fitc antibody
    Anti Kv11 1 Herg Extracellular Fitc Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti kv11 1 antibody  (Alomone Labs)


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    Alomone Labs anti kv11 1 antibody
    Anti Kv11 1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    kv11 1 apc 016  (Alomone Labs)


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    Alomone Labs kv11 1 apc 016
    I Kr currents and <t>Kv11.1</t> protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.
    Kv11 1 Apc 016, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy"

    Article Title: Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2022.942769

    I Kr currents and Kv11.1 protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.
    Figure Legend Snippet: I Kr currents and Kv11.1 protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.

    Techniques Used: Western Blot

    Nedd4-2-mediated ubiquitination of Kv11.1 was increased in pCH. (A) Representative immunoblots showing Nedd4-2, phosphorylated Nedd4-2 (p-Nedd4-2) and Rab11 proteins and corresponding quantifications of band densities in myocardium from control (CON) ( n = 5) and pCH (Ang II, n = 7). GADPH was used as an internal control to normalize the bands. (B) Representative blots immunoprecipitated (IP) by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-Nedd4-2 antibody. Quantification of band densities was shown as the ratio of Nedd4-2 to Kv11.1. (C) Representative blots immunoprecipitated by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-ubiquitin antibody. Quantification of band densities was shown as the ratio of ubiquitin to Kv11.1. (D) Proteins from control and pCH myocardium were immunoprecipitated with an anti-Kv7.1 or anti-IgG antibody, and IB was performed by using anti-Nedd4-2 antibody. IgG was used as negative controls. n = 3–4. * p < 0.05 and *** p < 0.001 versus CON. ns: not statistically significant.
    Figure Legend Snippet: Nedd4-2-mediated ubiquitination of Kv11.1 was increased in pCH. (A) Representative immunoblots showing Nedd4-2, phosphorylated Nedd4-2 (p-Nedd4-2) and Rab11 proteins and corresponding quantifications of band densities in myocardium from control (CON) ( n = 5) and pCH (Ang II, n = 7). GADPH was used as an internal control to normalize the bands. (B) Representative blots immunoprecipitated (IP) by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-Nedd4-2 antibody. Quantification of band densities was shown as the ratio of Nedd4-2 to Kv11.1. (C) Representative blots immunoprecipitated by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-ubiquitin antibody. Quantification of band densities was shown as the ratio of ubiquitin to Kv11.1. (D) Proteins from control and pCH myocardium were immunoprecipitated with an anti-Kv7.1 or anti-IgG antibody, and IB was performed by using anti-Nedd4-2 antibody. IgG was used as negative controls. n = 3–4. * p < 0.05 and *** p < 0.001 versus CON. ns: not statistically significant.

    Techniques Used: Western Blot, Immunoprecipitation

    Overexpression of mNedd4-2 prevented the downregulation of Kv11.1 channels. (A) Representative I Kr tail current tracings elicited by voltage pulses shown in the inset from isolated ventricular myocytes. (B) Quantification of I Kr currents density-voltage relationship ( n = 9–24 from 3 to 5 hearts in each group). (C) Representative immunoblots showed that left ventricular tissue proteins were pulled down by anti-Kv11.1 or anti-IgG antibodies and probed by anti-ubiquitin antibody ( n = 3). (D) Representative I Ks currents elicited by a pulse protocol shown in the inset. (E) Summary data of I Ks currents density-voltage relationship ( n = 14–30 from 3 to 5 hearts in each group). * p < 0.05 versus CON, # p < 0.05 versus Ang II.
    Figure Legend Snippet: Overexpression of mNedd4-2 prevented the downregulation of Kv11.1 channels. (A) Representative I Kr tail current tracings elicited by voltage pulses shown in the inset from isolated ventricular myocytes. (B) Quantification of I Kr currents density-voltage relationship ( n = 9–24 from 3 to 5 hearts in each group). (C) Representative immunoblots showed that left ventricular tissue proteins were pulled down by anti-Kv11.1 or anti-IgG antibodies and probed by anti-ubiquitin antibody ( n = 3). (D) Representative I Ks currents elicited by a pulse protocol shown in the inset. (E) Summary data of I Ks currents density-voltage relationship ( n = 14–30 from 3 to 5 hearts in each group). * p < 0.05 versus CON, # p < 0.05 versus Ang II.

    Techniques Used: Over Expression, Isolation, Western Blot

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    Alomone Labs kv11 1
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    Alomone Labs anti kv11 1 herg
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    Alomone Labs surface membrane kv11 1
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    Alomone Labs anti kv11 1 herg extracellular fitc antibody
    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for <t>Kv11.1-wt</t> and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.
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    Alomone Labs kv11 1 apc 016
    I Kr currents and <t>Kv11.1</t> protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.
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    Image Search Results


    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Mutagenesis, Binding Assay, Sequencing

    A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

    Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Dominant Negative Mutation, Construct, Transfection, Activation Assay

    Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Activation Assay

    The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

    Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Expressing, Transfection, Staining, Fluorescence

    A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Incubation, Western Blot, Cotransfection

    A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Article Snippet: After blocking, membranes were incubated with primary antibodies directed against the Kv11.1 C-terminus (APC-016, 1∶2000; Alomone, Jerusalem, Israel) or against the HA-epitope (H-9658, 1∶5000; Sigma) and secondary antibodies were horseradish peroxidase-conjugated (Jackson Immunoresearch, West Grove, PA).

    Techniques: Incubation, Expressing

    A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A: Located at the C-terminus, the P1086fs+32X (3256InsG) mutation is caused by a guanosine insertion in the codon at position 3256 (2356InsG), which elicits a frameshift at proline 1086 and produces 32 new amino acids before a premature stop codon. The mutation is downstream of the cyclic nucleotide binding domain (cNBD) and produces a truncated channel subunit. The N-terminus contains the Per Arnt Sim domain (PAS) and a HA-tag. B: Sequences for Kv11.1-wt and Kv11.1-mut (P1086fs+32X) including the nonsense 32 amino acid sequence.

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Mutagenesis, Binding Assay, Sequencing

    A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A: Immunoblot of equal amounts of protein lysates (25 µg) from HEK cells transfected with 1.0 or 2.0 µg of Kv11.1 cDNA. Kv11.1-wt channels expressed two protein bands corresponding to an immature core-glycosylated 135 kDa ER-resident Kv11.1 protein [wt-(I)], and a mature complex-glycosylated 155 kDa Kv11.1 band [wt-(M)]. Mutant Kv11.1 channels produced a single band at a slightly lower molecular weight (predicted to be 4 kDa smaller than Kv11.1-wt, thus approximately 131 kDa) corresponding to an immature core-glycosylated Kv11.1-mut protein [mut-(I)]. B: Densitometric analysis of total Kv11.1 protein (n = 4 experiments) demonstrated that Kv11.1-mut transfections resulted in significantly less total Kv11.1 protein expression than control or co-transfection (ANOVA *p<0.01). C,D: Reciprocal co-immunoprecipitation of Kv11.1-wt and Kv11.1-mut channels. Cells were transfected with a Kv11.1-wt construct lacking the HA-tag and Kv11.1-HA-mut. Co-immunoprecipitation was performed with anti-Kv11.1-wt antibody (C) (epitope corresponding to C-terminal 16 amino acids) or anti-HA antibody (D) for recognition of Kv11.1-mut. The two channel constructs strongly interacted. (Φ is a sample in which primary antibody was excluded during binding; IP: immunoprecipitation; IB: immunoblot).

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Western Blot, Transfection, Mutagenesis, Produced, Molecular Weight, Expressing, Cotransfection, Immunoprecipitation, Construct, Binding Assay

    Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: Electrophysiological properties of Kv11.1-wt and Kv11.1-mut channels were assessed using whole-cell patch clamping. A: Families of current tracings from −80 to +60 mV following 3 s step depolarizations. Kv11.1-wt currents were reduced following coexpression with Kv11.1-mut, indicating a dominant-negative suppression currents. Kv11.1-mut constructs were indistinguishable from GFP-transfected controls. B: The current-voltage relationship demonstrated that peak current amplitude is significantly reduced following coexpression (2.0 µg Kv11.1-wt, 57.7±4.6 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 51.4±6.3 pA/pF, n = 14; 1.0 µg Kv11.1-wt+1.0 µg Kv11.1-mut, 25.3±2.0 pA/pF, n = 10, p<0.001 from Kv11.1-wt). Peak Kv11.1-mut currents were similar to GFP-transfected cells (2.0 µg Kv11.1-mut, 6.5±0.8 pA/pF, n = 15 versus 0.25 µg GFP, 5.1±0.5 pA/pF, n = 5). The current-voltage profile and C-type inactivation properties were identical following normalization (inset). C: Peak tail currents were measured immediately following repolarization. Kv11.1-wt+Kv11.1-mut tails were significantly reduced compared to control (2.0 µg Kv11.1-wt, 52.8±2.8 pA/pF, n = 16; 1.0 µg Kv11.1-wt, 43.5±3.9 pA/pF, n = 14; 1.0 Kv11.1-wt+1.0 µg Kv11.1-mut, 25.9±2.6 pA/pF, n = 10; p<0.01 from Kv11.1-wt). Tail currents were normalized and fit to a Boltzmann function to assess the steady-state activation properties (inset). No changes in slope or V1/2 parameters were observed.

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Dominant Negative Mutation, Construct, Transfection, Activation Assay

    Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: Channel kinetics were compared between Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups as no appreciable currents could be measured from Kv11.1-mut alone. There was no difference in channel activation (A), deactivation (B), contribution of the fast component to current decay (C), steady-state inactivation (D), fast inactivation (E) or recovery from inactivation (F).

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Activation Assay

    The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: The staining patterns for cells co-transfected with GFP (green) and HA-tagged Kv11.1 plasmids (CY3, red) were assessed using immunocytochemistry and confocal microscopy. A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of both plasmids. Untransfected cells served as negative controls (D). DAPI stained nuclei (blue) and phalloidin stained actin filaments (CY5, purple) were used to identify the nucleus and plasma membrane, respectively. White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Scale bar represents 20 µm.

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Staining, Transfection, Immunocytochemistry, Confocal Microscopy, Expressing, Fluorescence

    Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: Mature Kv11.1 protein expression was investigated using an external Kv11.1 epitope (CY3, red). A: Kv11.1-wt; B: Kv11.1-mut; C: co-expression of Kv11.1-wt and Kv11.1-mut. GFP-transfected cells served as negative controls (D); DAPI stained nuclei (blue); phalloidin stained actin filaments (CY5, purple). White arrows indicate the location of line scans through the plasma membrane and perinuclear regions of merged images. Profile histograms indicate the fluorescence intensity for pixels along line scans for each group. Black arrows indicate the approximate location of plasma membrane in the histogram panels. Scale bar represents 10 µm.

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Expressing, Transfection, Staining, Fluorescence

    A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A/B: Cells were incubated at 30°C for 24 h and total Kv11.1 protein was assessed by Western blot. Reduced temperature did not change the intensity of the protein band nor cause the appearance of a Kv11.1-mut mature protein band. Co-transfection of non-HA-tagged Kv11.1-wt and HA-Kv11.1-mut (1.0 µg wt+1.0 µg HA-mut; in lanes 3 and 7) allowed for the specific identification of Kv11.1-mut protein (A; anti-HA antibody) and Kv11.1-wt protein (B; anti-Kv11.1 C-terminal antibody). C: Peak current-voltage relationship for Kv11.1-mut alone at 37°C and 30°C revealed no change in current density (Kv11.1-mut at 37°C, 6.5±0.8 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 8.8±0.9 pA/pF, n = 4). D: Peak tail current amplitude did not significantly change with reduced temperature (Kv11.1-mut at 37°C, −1.8±0.3 pA/pF, n = 15 versus Kv11.1-mut at 30°C, 2.1±2.0 pA/pF).

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Incubation, Western Blot, Cotransfection

    A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Journal: PLoS ONE

    Article Title: Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

    doi: 10.1371/journal.pone.0018273

    Figure Lengend Snippet: A: Incubation with the proteasomal inhibitor lactacystin (20 µM) for 24 h enhanced the expression of immature Kv11.1-mut protein, but did produce a complex-glycosylated Kv11.1-mut protein. B: Densitometric analysis of total protein expression after lactacystin treatment (+) normalized to non-treated lysates (−). There was a significant increase in the expression of total Kv11.1-mut protein compared to the other groups (ANOVA *p<0.01). Untreated Kv11.1-mut cells (2.0 µg Kv11.1-mut, 1.53±0.19, n = 5) versus 2.0 µg Kv11.1-wt control (0.80±0.05) and 1.0 ug Kv11.1-wt+1.0 µg Kv11.1-mut (0.80±0.10, n = 3). C: Twenty-four h treatment with the Kv11.1 channel blocker E-4031 (5 µM) enhanced the mature Kv11.1 protein band in Kv11.1-wt and Kv11.1-wt+Kv11.1-mut groups, but did not elicit a mature Kv11.1-mut channel. D: Combined 24 h treatment with lactacystin (20 µM) and E-4031 (5 µM) did not significantly enhance Kv11.1-mut protein expression, nor did it rescue channel maturation in the Kv11.1-mut or Kv11.1-wt+Kv11.1-mut groups.

    Article Snippet: For total Kv11.1 protein expression, cells were permeabilized with 0.5% Triton X-100 and probed with an anti-HA antibody (Sigma, H-2095), while total surface membrane Kv11.1 was probed in non-permeabilized cells with an anti-Kv11.1 antibody (Alomone, APC-109) that recognizes an extracellular 16-amino acid epitope located between S1 and S2.

    Techniques: Incubation, Expressing

    I Kr currents and Kv11.1 protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.

    Journal: Frontiers in Pharmacology

    Article Title: Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy

    doi: 10.3389/fphar.2022.942769

    Figure Lengend Snippet: I Kr currents and Kv11.1 protein were reduced in the myocardium in pCH. (A) Representative tail traces of I Kr before (left) and after application of E-4031 (2 μM) (right) in upper panel. Ventricular myocyte was pulsed as shown protocol. The lower panel showing representative tail traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (B) Summary data for I Kr tail current density-voltage relationship in control and pCH ( n = 10–14 cardiomyocytes from 3 to 5 hearts, * p < 0.05 versus CON). (C) Normalized current-voltage relationship for I Kr tail current. Curves were fit by Boltzmann function. (D) Representative I Ks traces recorded using the pulse protocol shown in the inset before (left) and after application of HMR1556 (2 μM) (right) in upper panel. The lower panel showing representative traces recorded in myocytes from control (CON) and pCH (Ang II), respectively. (E) Summary data for I Ks tail current density-voltage relationship in control and pCH ( n = 11–15 cardiomyocytes from 3 to 5 hearts). (F) Normalized current-voltage relationship for I Ks tail current. Curves were fit by Boltzmann function. (G) Representative immunoblots bands for mature and immature Kv11.1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). (H) Representative immunoblots bands for Kv7.1 and KCNE1 proteins and corresponding summary data (CON n = 5, Ang II n = 7). GADPH was used as an internal control to normalize these bands. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus CON. ns: not statistically significant.

    Article Snippet: Antibodies against Kv7.1 (APC-022), KCNE1 (APC-163), or Kv11.1 (APC-016) were obtained from Alomone Labs, Jerusalem, Israel.

    Techniques: Western Blot

    Nedd4-2-mediated ubiquitination of Kv11.1 was increased in pCH. (A) Representative immunoblots showing Nedd4-2, phosphorylated Nedd4-2 (p-Nedd4-2) and Rab11 proteins and corresponding quantifications of band densities in myocardium from control (CON) ( n = 5) and pCH (Ang II, n = 7). GADPH was used as an internal control to normalize the bands. (B) Representative blots immunoprecipitated (IP) by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-Nedd4-2 antibody. Quantification of band densities was shown as the ratio of Nedd4-2 to Kv11.1. (C) Representative blots immunoprecipitated by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-ubiquitin antibody. Quantification of band densities was shown as the ratio of ubiquitin to Kv11.1. (D) Proteins from control and pCH myocardium were immunoprecipitated with an anti-Kv7.1 or anti-IgG antibody, and IB was performed by using anti-Nedd4-2 antibody. IgG was used as negative controls. n = 3–4. * p < 0.05 and *** p < 0.001 versus CON. ns: not statistically significant.

    Journal: Frontiers in Pharmacology

    Article Title: Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy

    doi: 10.3389/fphar.2022.942769

    Figure Lengend Snippet: Nedd4-2-mediated ubiquitination of Kv11.1 was increased in pCH. (A) Representative immunoblots showing Nedd4-2, phosphorylated Nedd4-2 (p-Nedd4-2) and Rab11 proteins and corresponding quantifications of band densities in myocardium from control (CON) ( n = 5) and pCH (Ang II, n = 7). GADPH was used as an internal control to normalize the bands. (B) Representative blots immunoprecipitated (IP) by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-Nedd4-2 antibody. Quantification of band densities was shown as the ratio of Nedd4-2 to Kv11.1. (C) Representative blots immunoprecipitated by anti-Kv11.1, and western immunoblotting (IB) was performed by using anti-ubiquitin antibody. Quantification of band densities was shown as the ratio of ubiquitin to Kv11.1. (D) Proteins from control and pCH myocardium were immunoprecipitated with an anti-Kv7.1 or anti-IgG antibody, and IB was performed by using anti-Nedd4-2 antibody. IgG was used as negative controls. n = 3–4. * p < 0.05 and *** p < 0.001 versus CON. ns: not statistically significant.

    Article Snippet: Antibodies against Kv7.1 (APC-022), KCNE1 (APC-163), or Kv11.1 (APC-016) were obtained from Alomone Labs, Jerusalem, Israel.

    Techniques: Western Blot, Immunoprecipitation

    Overexpression of mNedd4-2 prevented the downregulation of Kv11.1 channels. (A) Representative I Kr tail current tracings elicited by voltage pulses shown in the inset from isolated ventricular myocytes. (B) Quantification of I Kr currents density-voltage relationship ( n = 9–24 from 3 to 5 hearts in each group). (C) Representative immunoblots showed that left ventricular tissue proteins were pulled down by anti-Kv11.1 or anti-IgG antibodies and probed by anti-ubiquitin antibody ( n = 3). (D) Representative I Ks currents elicited by a pulse protocol shown in the inset. (E) Summary data of I Ks currents density-voltage relationship ( n = 14–30 from 3 to 5 hearts in each group). * p < 0.05 versus CON, # p < 0.05 versus Ang II.

    Journal: Frontiers in Pharmacology

    Article Title: Pharmacological suppression of Nedd4-2 rescues the reduction of Kv11.1 channels in pathological cardiac hypertrophy

    doi: 10.3389/fphar.2022.942769

    Figure Lengend Snippet: Overexpression of mNedd4-2 prevented the downregulation of Kv11.1 channels. (A) Representative I Kr tail current tracings elicited by voltage pulses shown in the inset from isolated ventricular myocytes. (B) Quantification of I Kr currents density-voltage relationship ( n = 9–24 from 3 to 5 hearts in each group). (C) Representative immunoblots showed that left ventricular tissue proteins were pulled down by anti-Kv11.1 or anti-IgG antibodies and probed by anti-ubiquitin antibody ( n = 3). (D) Representative I Ks currents elicited by a pulse protocol shown in the inset. (E) Summary data of I Ks currents density-voltage relationship ( n = 14–30 from 3 to 5 hearts in each group). * p < 0.05 versus CON, # p < 0.05 versus Ang II.

    Article Snippet: Antibodies against Kv7.1 (APC-022), KCNE1 (APC-163), or Kv11.1 (APC-016) were obtained from Alomone Labs, Jerusalem, Israel.

    Techniques: Over Expression, Isolation, Western Blot