rabbit anti c terminal herg antibody  (Alomone Labs)


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    Alomone Labs rabbit anti c terminal herg antibody
    (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of <t>HERG</t> 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with <t>anti-C</t> terminal HERG antibody and were repeated twice (n = 2).
    Rabbit Anti C Terminal Herg Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti c terminal herg antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti c terminal herg antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel"

    Article Title: Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032654

    (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).
    Figure Legend Snippet: (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Techniques Used: Western Blot, Expressing, Mutagenesis

    (A) Western blot analysis of cell lysates from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone, HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Mature forms of HERG 1a cmyc and HERG 1b are indicated by 155 kDa and 95 kDa labels, respectively. Immature forms of HERG 1a cmyc and HERG 1b are indicated by 135 kDa and 85 kDa labels, respectively. Equal amounts of cell lysate were loaded in all lanes as assessed using BCA protein assay quantification Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2). (B) Western blot analysis of immunoprecipitations using anti-cmyc antibody from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone , HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).
    Figure Legend Snippet: (A) Western blot analysis of cell lysates from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone, HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Mature forms of HERG 1a cmyc and HERG 1b are indicated by 155 kDa and 95 kDa labels, respectively. Immature forms of HERG 1a cmyc and HERG 1b are indicated by 135 kDa and 85 kDa labels, respectively. Equal amounts of cell lysate were loaded in all lanes as assessed using BCA protein assay quantification Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2). (B) Western blot analysis of immunoprecipitations using anti-cmyc antibody from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone , HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Techniques Used: Western Blot, Transfection, Expressing, Mutagenesis, Bicinchoninic Acid Protein Assay

    (A) Representative western blot analysis of cell lysate from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown at 37°C and 27°C. (B) Representative western blot analysis of cell lysates from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown in the presence (+) or absence (−) of 10 µM E4031 for 36 hours. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).
    Figure Legend Snippet: (A) Representative western blot analysis of cell lysate from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown at 37°C and 27°C. (B) Representative western blot analysis of cell lysates from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown in the presence (+) or absence (−) of 10 µM E4031 for 36 hours. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Techniques Used: Western Blot, Expressing

    rabbit anti c terminal herg antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti c terminal herg antibody
    (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of <t>HERG</t> 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with <t>anti-C</t> terminal HERG antibody and were repeated twice (n = 2).
    Rabbit Anti C Terminal Herg Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti c terminal herg antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti c terminal herg antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel"

    Article Title: Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032654

    (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).
    Figure Legend Snippet: (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Techniques Used: Western Blot, Expressing, Mutagenesis

    (A) Western blot analysis of cell lysates from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone, HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Mature forms of HERG 1a cmyc and HERG 1b are indicated by 155 kDa and 95 kDa labels, respectively. Immature forms of HERG 1a cmyc and HERG 1b are indicated by 135 kDa and 85 kDa labels, respectively. Equal amounts of cell lysate were loaded in all lanes as assessed using BCA protein assay quantification Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2). (B) Western blot analysis of immunoprecipitations using anti-cmyc antibody from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone , HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).
    Figure Legend Snippet: (A) Western blot analysis of cell lysates from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone, HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Mature forms of HERG 1a cmyc and HERG 1b are indicated by 155 kDa and 95 kDa labels, respectively. Immature forms of HERG 1a cmyc and HERG 1b are indicated by 135 kDa and 85 kDa labels, respectively. Equal amounts of cell lysate were loaded in all lanes as assessed using BCA protein assay quantification Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2). (B) Western blot analysis of immunoprecipitations using anti-cmyc antibody from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone , HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Techniques Used: Western Blot, Transfection, Expressing, Mutagenesis, Bicinchoninic Acid Protein Assay

    (A) Representative western blot analysis of cell lysate from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown at 37°C and 27°C. (B) Representative western blot analysis of cell lysates from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown in the presence (+) or absence (−) of 10 µM E4031 for 36 hours. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).
    Figure Legend Snippet: (A) Representative western blot analysis of cell lysate from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown at 37°C and 27°C. (B) Representative western blot analysis of cell lysates from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown in the presence (+) or absence (−) of 10 µM E4031 for 36 hours. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Techniques Used: Western Blot, Expressing

    anti kcnh2  (Alomone Labs)


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    Alomone Labs anti kcnh2
    Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG <t>(KCNH2)</t> protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.
    Anti Kcnh2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kcnh2/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kcnh2 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging"

    Article Title: Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging

    Journal: Theranostics

    doi: 10.7150/thno.70884

    Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG (KCNH2) protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.
    Figure Legend Snippet: Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG (KCNH2) protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.

    Techniques Used: Expressing

    apc-109  (Alomone Labs)


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    Alomone Labs apc-109
    Apc 109, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    apc-109 - by Bioz Stars, 2023-02
    94/100 stars

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    herg pab  (Alomone Labs)


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    Alomone Labs herg pab
    Herg Pab, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    anti kv11 1 antibody  (Alomone Labs)


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    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: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kv11 1 antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kv11 1 antibody - by Bioz Stars, 2023-02
    94/100 stars

    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

    rabbit polyclonal anti herg  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti herg
    Rabbit Polyclonal Anti Herg, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit polyclonal anti herg antibody  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti herg antibody
    Rabbit Polyclonal Anti Herg Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    herg  (Alomone Labs)


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    Alomone Labs herg
    (A) Human ether-a-go-go-related gene <t>(hERG)</t> K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase <t>A2</t> <t>(EphA2)</t> protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Herg, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Herg K + Channel-Dependent Apoptosis and Cell Cycle Arrest in Human Glioblastoma Cells"

    Article Title: Herg K + Channel-Dependent Apoptosis and Cell Cycle Arrest in Human Glioblastoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088164

    (A) Human ether-a-go-go-related gene (hERG) K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase A2 (EphA2) protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: (A) Human ether-a-go-go-related gene (hERG) K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase A2 (EphA2) protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Expressing, Activation Assay, Western Blot

    rabbit anti herg primary polyclonal antibody  (Alomone Labs)


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    Alomone Labs rabbit anti herg primary polyclonal antibody
    Rabbit Anti Herg Primary Polyclonal Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit anti c terminal herg antibody
    (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of <t>HERG</t> 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with <t>anti-C</t> terminal HERG antibody and were repeated twice (n = 2).
    Rabbit Anti C Terminal Herg Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti kcnh2
    Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG <t>(KCNH2)</t> protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.
    Anti Kcnh2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs apc-109
    Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG <t>(KCNH2)</t> protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.
    Apc 109, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs herg pab
    Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG <t>(KCNH2)</t> protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.
    Herg Pab, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
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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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    (A) Human ether-a-go-go-related gene <t>(hERG)</t> K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase <t>A2</t> <t>(EphA2)</t> protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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    (A) Human ether-a-go-go-related gene <t>(hERG)</t> K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase <t>A2</t> <t>(EphA2)</t> protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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    Image Search Results


    (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Journal: PLoS ONE

    Article Title: Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

    doi: 10.1371/journal.pone.0032654

    Figure Lengend Snippet: (A) Western blot of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit. Mature fully-glycosylated HERG 1a subunit (indicated by 155 kDa label) and immature HERG 1a subunit (indicated by 135 kDa label) are indicated. (B) Western blots of membrane protein extracts from stable HEK293 cell lines expressing full-length WT or LQT2 mutant forms of HERG 1a channel subunit before (−) and after treatment (+) with N-glycosidase F. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Article Snippet: Proteins were transferred by semi-dry blotting to a nitrocellulose membrane and incubated overnight at 4°C with a rabbit anti-C terminal HERG antibody (Alomone Labs) and detected by ECL.

    Techniques: Western Blot, Expressing, Mutagenesis

    (A) Western blot analysis of cell lysates from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone, HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Mature forms of HERG 1a cmyc and HERG 1b are indicated by 155 kDa and 95 kDa labels, respectively. Immature forms of HERG 1a cmyc and HERG 1b are indicated by 135 kDa and 85 kDa labels, respectively. Equal amounts of cell lysate were loaded in all lanes as assessed using BCA protein assay quantification Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2). (B) Western blot analysis of immunoprecipitations using anti-cmyc antibody from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone , HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Journal: PLoS ONE

    Article Title: Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

    doi: 10.1371/journal.pone.0032654

    Figure Lengend Snippet: (A) Western blot analysis of cell lysates from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone, HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Mature forms of HERG 1a cmyc and HERG 1b are indicated by 155 kDa and 95 kDa labels, respectively. Immature forms of HERG 1a cmyc and HERG 1b are indicated by 135 kDa and 85 kDa labels, respectively. Equal amounts of cell lysate were loaded in all lanes as assessed using BCA protein assay quantification Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2). (B) Western blot analysis of immunoprecipitations using anti-cmyc antibody from transiently transfected HEK293 cells co-expressing WT HERG 1a cmyc tagged subunit alone, WT HERG 1b untagged alone , HERG 1a cmyc with HERG 1b or LQT2 mutant HERG 1a cmyc with HERG 1b. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Article Snippet: Proteins were transferred by semi-dry blotting to a nitrocellulose membrane and incubated overnight at 4°C with a rabbit anti-C terminal HERG antibody (Alomone Labs) and detected by ECL.

    Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Bicinchoninic Acid Protein Assay

    (A) Representative western blot analysis of cell lysate from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown at 37°C and 27°C. (B) Representative western blot analysis of cell lysates from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown in the presence (+) or absence (−) of 10 µM E4031 for 36 hours. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Journal: PLoS ONE

    Article Title: Changes in Channel Trafficking and Protein Stability Caused by LQT2 Mutations in the PAS Domain of the HERG Channel

    doi: 10.1371/journal.pone.0032654

    Figure Lengend Snippet: (A) Representative western blot analysis of cell lysate from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown at 37°C and 27°C. (B) Representative western blot analysis of cell lysates from stable HEK293 cells expressing WT and traffic deficient LQT2 mutations in the HERG 1a subunit grown in the presence (+) or absence (−) of 10 µM E4031 for 36 hours. Western blots were probed with anti-C terminal HERG antibody and were repeated twice (n = 2).

    Article Snippet: Proteins were transferred by semi-dry blotting to a nitrocellulose membrane and incubated overnight at 4°C with a rabbit anti-C terminal HERG antibody (Alomone Labs) and detected by ECL.

    Techniques: Western Blot, Expressing

    Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG (KCNH2) protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.

    Journal: Theranostics

    Article Title: Stress-induced premature senescence is associated with a prolonged QT interval and recapitulates features of cardiac aging

    doi: 10.7150/thno.70884

    Figure Lengend Snippet: Alterations of I Kr , I NaL and Ca 2+ handling in SenCMs. (A) E-4031 (3µM)-sensitive currents (I Kr ) activated according to the protocol shown on top and relative I/V relationships in iCMs (N = 21) and SenCMs (N = 26) 5-7 days after treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (B) HERG (KCNH2) protein expression levels in iCMs and SenCMs. Quantitative data of four independent experiments ± SEM (densitometric values for the protein of interest normalized for histone H3). ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (C) TTX (2 µM)-sensitive current (I TTX ) activated during slow voltage ramps (28 mV/sec) from a holding potential of -100 mV. Mean ± SEM I/V relationships for iCMs (N = 12) and SenCMs (N = 13) are shown. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. Statistics of I TTX at 0 mV, representative of I NaL , are reported on the right. (D) pThr286 CAMKII:CAMKII total protein expression levels in SenCMs versus iCMs. Quantitative data are from four independent experiments ± SEM (densitometric values for the proteins of interest normalized for GAPDH). * P < 0.05, ** P < 0.01 vs iCMs. (E) Membrane currents and Ca 2+ transients (CaT) were recorded simultaneously according to the voltage clamp protocol shown on top in Fluo4-loaded iCMs. Examples (left panel) and statistics (right panel) of CaL influx, CaT amplitude and caffeine-induced CaT amplitude (estimating CaSR) in iCMs (N = 22) and SenCMs (N= 14) 5-7 days after Dox treatment. Data pooled from four independent experiments are presented as ± SEM. * P < 0.05, ** P < 0.01 vs iCMs. (F) SERCA2 and monomeric (m) PLN protein expression levels in iCMs and SenCMs. Quantitative data of five independent experiments ± SEM (densitometric values for the protein of interest normalized for actin). (G) Statistics of Ca 2+ spark rate and characteristics in SenCMs (N=410) vs iCMs (N=266). Line scan (xt) images are shown on the left (time bar: 100 ms). * P < 0.05, ** P < 0.01 vs iCMs.

    Article Snippet: The membranes were blocked for 1 h with Intercept (TBS) Blocking Buffer (Licor) or milk and incubated with the primary Abs at 4 °C overnight (anti-p16, 1:1'000, Proteintech 10883; anti-p21, 1:1'000 eBiosciences 14-6715-81; anti-KCNH2, 1:200, Alomone APC-109; anti-P AMPK, 1:1'000, Cell Signaling 2537; anti-CAMKII, 1:2'000, Abcam 92332; anti-P CAMKII, 1:2'000, Abcam 171095; anti-SERCA2, 1:1000, N-19 Santa Cruz Biotechnology; anti-PLN 1:1000, 2D12, Abcam; anti-actin 1:5000, Merck).

    Techniques: 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

    (A) Human ether-a-go-go-related gene (hERG) K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase A2 (EphA2) protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Journal: PLoS ONE

    Article Title: Herg K + Channel-Dependent Apoptosis and Cell Cycle Arrest in Human Glioblastoma Cells

    doi: 10.1371/journal.pone.0088164

    Figure Lengend Snippet: (A) Human ether-a-go-go-related gene (hERG) K + channels exhibited reduced expression in the presence of doxazosin. (B) Doxazosin-induced apoptosis was further associated with lower erythropoietin-producing human hepatocellular carcinoma receptor tyrosine kinase A2 (EphA2) protein levels. Enhanced phosphorylation (i.e. activation) of EphA2 (C, normalized p-EphA2 levels; D, p-EphA2 relative to total EphA2) and of p38 mitogen-activated protein kinase (MAPK; F, normalized p-p38 MAPK levels; G, p-p38 MAPK relative to total p38 MAPK) was observed, while total p38 MAPK levels were not significantly affected by doxazosin (E). Finally, growth arrest and DNA damage inducible gene 153 (GADD153) protein levels were elevated upon doxazosin treatment (H). Original Western Blots and mean data normalized to control conditions obtained from n = 3 independent assays (± SEM) are provided (*p<0.05; **p<0.01). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Article Snippet: Nitrocellulose membranes were developed by sequential exposure to blocking reagent (5% dry milk), primary antibodies directed against hERG (1∶200; APC-016, Alomone Labs, Jerusalem, Israel), EphA2 (1∶100; sc-924, Santa Cruz Biotechnology, Heidelberg, Germany), phospho-EphA2/Tyr-593 (1∶1,000; CB4368, Cell Applications, San Diego, CA, USA), growth arrest and DNA damage inducible gene 153 (GADD153; 1∶500; ab11419, Abcam), p38 mitogen-activated protein kinase (MAPK; 1∶1,000; 9212, Cell Signaling), phospho-p38 MAPK/Thr-180/Tyr-182 (1∶1,000; 9211, Cell Signaling), caspase 3 (1∶1,000; 9662, Cell Signaling), cleaved caspase 3 (1∶1,000; 9664, Cell Signaling), caspase 7 (1∶1,000; 9492, Cell Signaling), cleaved caspase 7 (1∶1,000; 9491, Cell Signaling), caspase 9 (1∶1,000; ab32539, Abcam), cleaved caspase 9 (1∶1,000; ab2324, Abcam), microtubule-associated protein 1 light chain 3 (LC3)A/B (1∶1,000; 4108, Cell Signaling), cleaved poly-ADP-ribose-polymerase (PARP; 1∶1000; 5625, Cell Signaling), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1∶40,000; G8140-11, US Biological, Swampscott, MA, USA), and appropriate horseradish peroxidase-conjugated secondary antibodies (Abcam).

    Techniques: Expressing, Activation Assay, Western Blot