anti kcne1 isk antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs anti kcne1 isk antibody
    Cleavage of K v 7.1 in physiology and pathophysiology. A , Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both co-expressed with <t>KCNE1.</t> B , Mean currents amplitude was plotted versus voltage to obtain current-voltage (I-V) relationships in cells expressing K v 7.1-MYC or K v 7.1-D459A-MYC and KCNE1 treated with 500 nmol/L staurosporine for 10 – 12 hours. Statistics were tested with Two-Way ANOVA followed by Bonferroni posttests. C , Western blot analysis of cells used for patch-clamp recordings. D , Schematic illustration to highlight the position of G460 and calmodulin binding site in helix A (upper left panel). Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A transfected cells served as negative controls (lower left panel). Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with One-Way-ANOVA followed by Bonferroni’s Multiple Comparison test. (right panel). (C) anti-K v 7.1 antibody, anti-KCNE1 antibody. (D) anti-MYC antibody, anti-β-actin antibody.
    Anti Kcne1 Isk Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 18 article reviews
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    anti kcne1 isk antibody - by Bioz Stars, 2022-08
    90/100 stars

    Images

    1) Product Images from "Doxorubicin induces caspase-mediated proteolysis of KV7.1"

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    Journal: bioRxiv

    doi: 10.1101/259242

    Cleavage of K v 7.1 in physiology and pathophysiology. A , Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both co-expressed with KCNE1. B , Mean currents amplitude was plotted versus voltage to obtain current-voltage (I-V) relationships in cells expressing K v 7.1-MYC or K v 7.1-D459A-MYC and KCNE1 treated with 500 nmol/L staurosporine for 10 – 12 hours. Statistics were tested with Two-Way ANOVA followed by Bonferroni posttests. C , Western blot analysis of cells used for patch-clamp recordings. D , Schematic illustration to highlight the position of G460 and calmodulin binding site in helix A (upper left panel). Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A transfected cells served as negative controls (lower left panel). Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with One-Way-ANOVA followed by Bonferroni’s Multiple Comparison test. (right panel). (C) anti-K v 7.1 antibody, anti-KCNE1 antibody. (D) anti-MYC antibody, anti-β-actin antibody.
    Figure Legend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. A , Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both co-expressed with KCNE1. B , Mean currents amplitude was plotted versus voltage to obtain current-voltage (I-V) relationships in cells expressing K v 7.1-MYC or K v 7.1-D459A-MYC and KCNE1 treated with 500 nmol/L staurosporine for 10 – 12 hours. Statistics were tested with Two-Way ANOVA followed by Bonferroni posttests. C , Western blot analysis of cells used for patch-clamp recordings. D , Schematic illustration to highlight the position of G460 and calmodulin binding site in helix A (upper left panel). Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A transfected cells served as negative controls (lower left panel). Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with One-Way-ANOVA followed by Bonferroni’s Multiple Comparison test. (right panel). (C) anti-K v 7.1 antibody, anti-KCNE1 antibody. (D) anti-MYC antibody, anti-β-actin antibody.

    Techniques Used: Expressing, Western Blot, Patch Clamp, Binding Assay, Construct, Transfection

    2) Product Images from "Doxorubicin induces caspase-mediated proteolysis of KV7.1"

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0162-z

    Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM
    Figure Legend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM

    Techniques Used: Expressing, Western Blot, Immunoprecipitation, Binding Assay, Construct, Transfection, Mutagenesis, Negative Control

    3) Product Images from "KCNQ1/KCNE1 ASSEMBLY, CO-TRANSLATION NOT REQUIRED"

    Article Title: KCNQ1/KCNE1 ASSEMBLY, CO-TRANSLATION NOT REQUIRED

    Journal: Channels (Austin, Tex.)

    doi:

    Inhibition of protein synthesis does not prevent prKCNE1 assembly with KCNQ1. (A) Whole-cell currents recorded at +50mV from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as a percentage of the current recorded prior to CHX (50 μg/ml) or EtOH treatment (N ≥ 5 for all time points). Arrow indicates when CHX or EtOH was added. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or CHX (bottom traces) for 6h, and then injected with prKCNE1 and further incubated in the presence of CHX or EtOH for an additional 8h. (C) Average currents recorded at +50mV (left panel) and average V ½ values (right panel) obtained from the EtOH-treated (open bar, N = 9) or CHX-treated (solid bar, N = 11) oocytes illustrated in Fig. 2B.
    Figure Legend Snippet: Inhibition of protein synthesis does not prevent prKCNE1 assembly with KCNQ1. (A) Whole-cell currents recorded at +50mV from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as a percentage of the current recorded prior to CHX (50 μg/ml) or EtOH treatment (N ≥ 5 for all time points). Arrow indicates when CHX or EtOH was added. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or CHX (bottom traces) for 6h, and then injected with prKCNE1 and further incubated in the presence of CHX or EtOH for an additional 8h. (C) Average currents recorded at +50mV (left panel) and average V ½ values (right panel) obtained from the EtOH-treated (open bar, N = 9) or CHX-treated (solid bar, N = 11) oocytes illustrated in Fig. 2B.

    Techniques Used: Inhibition, Injection, Expressing, Incubation

    Time-course for prKCNE1 modulation of KCNQ1 channels. (A) Schematic showing injection/incubation protocol. (B) Representative current traces recorded from KCNQ1-expressing oocytes at 2, 4, 6 and 18h following prKCNE1 injection. (C) V ½ values for whole-cell currents recorded from oocytes injected with KCNQ1 cRNA (open bar) and KCNQ1 cRNA and prKCNE1 (solid bars). The dashed gray line denotes the V ½ obtained for KCNQ1-KCNE1 channels translated by the oocytes. (D) Time-course for prKCNE1 functional interaction with KCNQ1. In this panel, the dashed line indicates ΔV ½ between KCNQ1-only and KCNQ1-KCNE1 channels translated by the oocytes. N: KCNQ1 cRNA = 9; KCNQ1 + KCNE1 cRNA = 13; KCNQ1 + prKCNE1, 2h = 3; 4h = 5; 6h = 12; 18h = 11; 24h = 8.
    Figure Legend Snippet: Time-course for prKCNE1 modulation of KCNQ1 channels. (A) Schematic showing injection/incubation protocol. (B) Representative current traces recorded from KCNQ1-expressing oocytes at 2, 4, 6 and 18h following prKCNE1 injection. (C) V ½ values for whole-cell currents recorded from oocytes injected with KCNQ1 cRNA (open bar) and KCNQ1 cRNA and prKCNE1 (solid bars). The dashed gray line denotes the V ½ obtained for KCNQ1-KCNE1 channels translated by the oocytes. (D) Time-course for prKCNE1 functional interaction with KCNQ1. In this panel, the dashed line indicates ΔV ½ between KCNQ1-only and KCNQ1-KCNE1 channels translated by the oocytes. N: KCNQ1 cRNA = 9; KCNQ1 + KCNE1 cRNA = 13; KCNQ1 + prKCNE1, 2h = 3; 4h = 5; 6h = 12; 18h = 11; 24h = 8.

    Techniques Used: Injection, Incubation, Expressing, Functional Assay

    Modulation of KCNQ1 by prKCNE1 requires vesicular trafficking. (A) Whole-cell currents recorded at +50mV following BFA (5 μM) or EtOH treatment from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as percentage of the current recorded prior to treatment (N ≥ 5 for all time points. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or BFA (bottom traces) for 3h, then injected with prKCNE1 and further incubated for 6 or 15h. (C) V ½ calculated from the normalized current data recorded after 6 and 15h incubations. The dashed line in the bottom panel is the V ½ calculated for KCNQ1-only channels and the open symbols are the V ½ values for currents recorded from KCNQ1-expressing oocytes 6 and 15h after prKCNE1 injection under control conditions. N 6 for both conditions at each time point.
    Figure Legend Snippet: Modulation of KCNQ1 by prKCNE1 requires vesicular trafficking. (A) Whole-cell currents recorded at +50mV following BFA (5 μM) or EtOH treatment from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as percentage of the current recorded prior to treatment (N ≥ 5 for all time points. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or BFA (bottom traces) for 3h, then injected with prKCNE1 and further incubated for 6 or 15h. (C) V ½ calculated from the normalized current data recorded after 6 and 15h incubations. The dashed line in the bottom panel is the V ½ calculated for KCNQ1-only channels and the open symbols are the V ½ values for currents recorded from KCNQ1-expressing oocytes 6 and 15h after prKCNE1 injection under control conditions. N 6 for both conditions at each time point.

    Techniques Used: Injection, Expressing, Incubation

    Intracellular processing of prKCNE1. (A) Average whole-cell currents from oocytes expressing KCNQ1 alone (N = 8) and those injected with prKCNE1 (N = 7) or prKCNE1-L51H (N = 10). (B) Average current-voltage relationships from oocytes injected with water (▽, N = 8), KCNQ1 cRNA (○, N = 8) or KCNQ1 cRNA + prKCNE1-L51H (◆, N = 10). Injection of KCNE1-L51H reduced KCNQ1 currents by ~40% when compared to LMPG injection. * = P
    Figure Legend Snippet: Intracellular processing of prKCNE1. (A) Average whole-cell currents from oocytes expressing KCNQ1 alone (N = 8) and those injected with prKCNE1 (N = 7) or prKCNE1-L51H (N = 10). (B) Average current-voltage relationships from oocytes injected with water (▽, N = 8), KCNQ1 cRNA (○, N = 8) or KCNQ1 cRNA + prKCNE1-L51H (◆, N = 10). Injection of KCNE1-L51H reduced KCNQ1 currents by ~40% when compared to LMPG injection. * = P

    Techniques Used: Expressing, Injection

    4) Product Images from "Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2"

    Article Title: Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.147033

    P127T LQT5 mutation and Δ109–129 deletion at the KCNE1 distal C-terminus suppress the cAMP-mediated upregulation of I KS current. Representative current traces recorded from CHO cells co-expressing WT KCNQ1+WT yotiao and WT KCNE1 (A), KCNE1 P127T (C), KCNE1 Δ109–129 (E) and KCNE1 V109I (G). Cells were held at −90 mV and stepped to +30 mV for 3 s and then repolarized to −40 mV for 1.5 s. Cells were recorded in the absence (black traces) or presence (red traces) of 200 µM cAMP+0.2 µM okadaic acid. Current–voltage relationships of WT KCNQ1+WT yotiao and WT KCNE1 (B) or KCNE1 P127T (D) or KCNE1 Δ109–129 (F) or KCNE1 V109I (H). Voltage was stepped for 3 s from −50 mV to +60 mV in 10 mV increments followed by repolarization to −40 mV for 1.5 s. Red and black curves represent recordings with or without cAMP+okadaic acid in the patch pipette, respectively. ( n = 6–7). Above +30 mV, the WT I KS and, above +20 mV, the KCNE1 V109I current upregulation induced by cAMP+okadaic acid were significant. * P
    Figure Legend Snippet: P127T LQT5 mutation and Δ109–129 deletion at the KCNE1 distal C-terminus suppress the cAMP-mediated upregulation of I KS current. Representative current traces recorded from CHO cells co-expressing WT KCNQ1+WT yotiao and WT KCNE1 (A), KCNE1 P127T (C), KCNE1 Δ109–129 (E) and KCNE1 V109I (G). Cells were held at −90 mV and stepped to +30 mV for 3 s and then repolarized to −40 mV for 1.5 s. Cells were recorded in the absence (black traces) or presence (red traces) of 200 µM cAMP+0.2 µM okadaic acid. Current–voltage relationships of WT KCNQ1+WT yotiao and WT KCNE1 (B) or KCNE1 P127T (D) or KCNE1 Δ109–129 (F) or KCNE1 V109I (H). Voltage was stepped for 3 s from −50 mV to +60 mV in 10 mV increments followed by repolarization to −40 mV for 1.5 s. Red and black curves represent recordings with or without cAMP+okadaic acid in the patch pipette, respectively. ( n = 6–7). Above +30 mV, the WT I KS and, above +20 mV, the KCNE1 V109I current upregulation induced by cAMP+okadaic acid were significant. * P

    Techniques Used: Mutagenesis, Expressing, Transferring

    Effects of KCNE1 C-terminus deletions and LQT5 mutations on I KS currents. (A) A cartoon of KCNE1, indicating the location of the LQT5 mutations and the deletions in the C-terminus. Representative current traces of WT KCNQ1, co-expressed in CHO cells with WT KCNE1 (B), LQT5 mutant V109I (C), LQT5 mutant P127T (D) or with KCNE1 deletion mutants at the distal C-terminus Δ109–129 (E) or the proximal C-terminus Δ69–77 (F). Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −50 mV to +60 mV (or to +100 mV for Δ69–77) in 10 mV increments and then repolarized for 1.5 s to −60 mV. Current–voltage (G) and conductance–voltage (H) relationships of WT KCNQ1+WT KCNE1 or LQT5 mutants ( n = 11–17). Current–voltage (I) and conductance–voltage (J) relationships of WT KCNQ1+WT KCNE1 or deletion mutants ( n = 7–10).
    Figure Legend Snippet: Effects of KCNE1 C-terminus deletions and LQT5 mutations on I KS currents. (A) A cartoon of KCNE1, indicating the location of the LQT5 mutations and the deletions in the C-terminus. Representative current traces of WT KCNQ1, co-expressed in CHO cells with WT KCNE1 (B), LQT5 mutant V109I (C), LQT5 mutant P127T (D) or with KCNE1 deletion mutants at the distal C-terminus Δ109–129 (E) or the proximal C-terminus Δ69–77 (F). Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −50 mV to +60 mV (or to +100 mV for Δ69–77) in 10 mV increments and then repolarized for 1.5 s to −60 mV. Current–voltage (G) and conductance–voltage (H) relationships of WT KCNQ1+WT KCNE1 or LQT5 mutants ( n = 11–17). Current–voltage (I) and conductance–voltage (J) relationships of WT KCNQ1+WT KCNE1 or deletion mutants ( n = 7–10).

    Techniques Used: Mutagenesis

    LQT1 and LQT5 mutations disrupt the interaction between KCNQ1 helix C and KCNE1 C-terminus. (A) Representative immunoblot of GST pulldown (GST PD) (upper row) of the His-MBP-tagged KCNE1 C-terminus (residues 67–129, E1CT in diagram) or control His-MBP peptide, by GST-tagged KCNQ1 helix C (residues 535–572, Q1 Helix C, in diagram), illustrating the direct interaction of KCNE1 C-terminus with KCNQ1 helix C ( n = 4). Equal amounts of His-MBP and His-MBP-tagged KCNE1 were used, as seen in the input (lower row). (B) Representative immunoblot of GST pulldown (upper row) of the His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504, Q1CT in diagram) by GST-tagged KCNE1 C-terminus deletion mutants (E1CT in diagram). Inputs are shown in the lower row. (C) Quantification of the pulldowns normalized to input ( n = 5). * P
    Figure Legend Snippet: LQT1 and LQT5 mutations disrupt the interaction between KCNQ1 helix C and KCNE1 C-terminus. (A) Representative immunoblot of GST pulldown (GST PD) (upper row) of the His-MBP-tagged KCNE1 C-terminus (residues 67–129, E1CT in diagram) or control His-MBP peptide, by GST-tagged KCNQ1 helix C (residues 535–572, Q1 Helix C, in diagram), illustrating the direct interaction of KCNE1 C-terminus with KCNQ1 helix C ( n = 4). Equal amounts of His-MBP and His-MBP-tagged KCNE1 were used, as seen in the input (lower row). (B) Representative immunoblot of GST pulldown (upper row) of the His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504, Q1CT in diagram) by GST-tagged KCNE1 C-terminus deletion mutants (E1CT in diagram). Inputs are shown in the lower row. (C) Quantification of the pulldowns normalized to input ( n = 5). * P

    Techniques Used:

    LQT5 P127T mutant and deletion mutant Δ109–129 impair phosphorylation of KCNQ1 at S27 during cAMP-dependent stimulation. (A) Representative immunoblots (IB) of HEK 293 cells lysates co-expressing yotiao, WT KCNQ1 and WT, mutated (V109I and P127T) or truncated (Δ109–129) KCNE1 from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid and immunoblots of HEK 293 cells lysates co-expressing yotiao WT KCNE1 and WT KCNQ1 or LQT1 mutant K557E from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid. Blots were probed with antibodies against phosphorylated KCNQ1 S27 (first row from top), KCNQ1 (second row from top), yotiao (third row from top) and KCNE1 (fourth row from top). (B) Quantification of phosphorylated KCNQ1 S27 was calculated by dividing phosphorylated signal to KCNQ1 input (anti-KCNQ1 blot) for both unstimulated and cAMP-stimulated cells (250 µM 8CPT+0.2 µM okadaic acid) and normalized to stimulated WT KCNQ1+WT KCNE1+yotiao ( n = 3–5). * P
    Figure Legend Snippet: LQT5 P127T mutant and deletion mutant Δ109–129 impair phosphorylation of KCNQ1 at S27 during cAMP-dependent stimulation. (A) Representative immunoblots (IB) of HEK 293 cells lysates co-expressing yotiao, WT KCNQ1 and WT, mutated (V109I and P127T) or truncated (Δ109–129) KCNE1 from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid and immunoblots of HEK 293 cells lysates co-expressing yotiao WT KCNE1 and WT KCNQ1 or LQT1 mutant K557E from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid. Blots were probed with antibodies against phosphorylated KCNQ1 S27 (first row from top), KCNQ1 (second row from top), yotiao (third row from top) and KCNE1 (fourth row from top). (B) Quantification of phosphorylated KCNQ1 S27 was calculated by dividing phosphorylated signal to KCNQ1 input (anti-KCNQ1 blot) for both unstimulated and cAMP-stimulated cells (250 µM 8CPT+0.2 µM okadaic acid) and normalized to stimulated WT KCNQ1+WT KCNE1+yotiao ( n = 3–5). * P

    Techniques Used: Mutagenesis, Western Blot, Expressing

    LQT1 and LQT5 mutations do not affect the channel trafficking to the plasma membrane. (A) TIRF fluorescence images of CHO cells co-expressing CFP-tagged WT KCNQ1 and GPI-citrine. (B) Epi-fluorescence (left panels) and TIRF fluorescence (right panels) images of CHO cells expressing CFP-tagged WT KCNQ1+KCNE1 (upper panels) or CFP-tagged G589D LQT1 mutant+KCNE1 (lower panels). (C) TIRF fluorescence images of CHO cells co-expressing CFP-tagged helix C LQT1 mutants with KCNE1. (D) YFP-tagged WT KCNE1 or LQT5 mutant co-expressed with WT KCNQ1. Quantification of the TIRF fluorescent signals of CFP-tagged WT KCNQ1 and LQT1 mutants (E) and of YFP-tagged WT and LQT5 mutant (F) as normalized to membrane signal area ( n = 16–60). *** P
    Figure Legend Snippet: LQT1 and LQT5 mutations do not affect the channel trafficking to the plasma membrane. (A) TIRF fluorescence images of CHO cells co-expressing CFP-tagged WT KCNQ1 and GPI-citrine. (B) Epi-fluorescence (left panels) and TIRF fluorescence (right panels) images of CHO cells expressing CFP-tagged WT KCNQ1+KCNE1 (upper panels) or CFP-tagged G589D LQT1 mutant+KCNE1 (lower panels). (C) TIRF fluorescence images of CHO cells co-expressing CFP-tagged helix C LQT1 mutants with KCNE1. (D) YFP-tagged WT KCNE1 or LQT5 mutant co-expressed with WT KCNQ1. Quantification of the TIRF fluorescent signals of CFP-tagged WT KCNQ1 and LQT1 mutants (E) and of YFP-tagged WT and LQT5 mutant (F) as normalized to membrane signal area ( n = 16–60). *** P

    Techniques Used: Fluorescence, Expressing, Mutagenesis

    5) Product Images from "BACE1 and presenilin/?-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels"

    Article Title: BACE1 and presenilin/?-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels

    Journal: The FASEB Journal

    doi: 10.1096/fj.12-214056

    Putative BACE1 and PS/γ-secretase cleavage sites of human KCNE1 and KCNE2.
    Figure Legend Snippet: Putative BACE1 and PS/γ-secretase cleavage sites of human KCNE1 and KCNE2.

    Techniques Used:

    Elevated BACE1 activity increases KCNE1- and KCNE2-CTF levels in B104 rat neuroblastoma cells. A ) Overexpression of human BACE1 increased KCNE1-CTF levels in B104 cells. DAPT treatment further enhanced KCNE1-CTF levels in control and BACE1-overexpressing cells. B ) KCNE2-CTF levels were also elevated by BACE1 overexpression in B104 cells.
    Figure Legend Snippet: Elevated BACE1 activity increases KCNE1- and KCNE2-CTF levels in B104 rat neuroblastoma cells. A ) Overexpression of human BACE1 increased KCNE1-CTF levels in B104 cells. DAPT treatment further enhanced KCNE1-CTF levels in control and BACE1-overexpressing cells. B ) KCNE2-CTF levels were also elevated by BACE1 overexpression in B104 cells.

    Techniques Used: Activity Assay, Over Expression

    BACE1 and PS/γ-secretase activities regulate endogenous KCNE1-CTF levels. A ) Detection of endogenous KCNE1 full-length (F.L.) and CTF bands in cultured mouse cardiomyocytes. DAPT (5 μM) treatment increased endogenous KCNE1-CTF levels. B ) Endogenous KCNE1-CTF levels were also increased by BACE1 overexpression in mouse cardiomyocytes.
    Figure Legend Snippet: BACE1 and PS/γ-secretase activities regulate endogenous KCNE1-CTF levels. A ) Detection of endogenous KCNE1 full-length (F.L.) and CTF bands in cultured mouse cardiomyocytes. DAPT (5 μM) treatment increased endogenous KCNE1-CTF levels. B ) Endogenous KCNE1-CTF levels were also increased by BACE1 overexpression in mouse cardiomyocytes.

    Techniques Used: Cell Culture, Over Expression

    PS/γ-secretase activity regulates generation of KCNE1- and KCNE2-ICDs. A ) Western blot analysis showed that KCNE1-ICD is specifically regulated by PS/γ-secretase activity in B104 cells stably expressing full-length KCNE1. Epoxomicin, a proteasome inhibitor, prevented KCNE1-ICD degradation. B ) KCNE2-ICD is detected in the similar conditions.
    Figure Legend Snippet: PS/γ-secretase activity regulates generation of KCNE1- and KCNE2-ICDs. A ) Western blot analysis showed that KCNE1-ICD is specifically regulated by PS/γ-secretase activity in B104 cells stably expressing full-length KCNE1. Epoxomicin, a proteasome inhibitor, prevented KCNE1-ICD degradation. B ) KCNE2-ICD is detected in the similar conditions.

    Techniques Used: Activity Assay, Western Blot, Stable Transfection, Expressing

    KCNE1 and KCNE2 undergo sequential cleavage mediated by α- and PS/γ-secretases. A ) Schematic diagram showing sequential cleavage of KCNE1 and KCNE2 by BACE1, α-, and PS/γ-secretases. α-Secretase inhibitor (TAPI-1) or BACE1 inhibitor ( D R9) decreases generation of KCNE C-terminal fragments (KCNE-CTFs), while PMA, an α-secretase activator, increases KCNE-CTF levels. KCNE-CTFs are then cleaved by PS/γ-secretases to generate KCNE intracellular domains (KCNE-ICDs). B ) Western blot analysis of human KCNE1 full-length (F.L.) and its CTF expressed in B104 cells. KCNE1-CTF levels were increased by treatment with DAPT and further elevated by cotreatment with PMA, while partially decreased by TAPI-1. C ) Western blot analysis of human KCNE2 F.L. and its CTF expressed in B104 cells. Similar to KCNE1, KCNE2-CTF levels are also increased by DAPT treatment and further elevated by cotreatment with PMA.
    Figure Legend Snippet: KCNE1 and KCNE2 undergo sequential cleavage mediated by α- and PS/γ-secretases. A ) Schematic diagram showing sequential cleavage of KCNE1 and KCNE2 by BACE1, α-, and PS/γ-secretases. α-Secretase inhibitor (TAPI-1) or BACE1 inhibitor ( D R9) decreases generation of KCNE C-terminal fragments (KCNE-CTFs), while PMA, an α-secretase activator, increases KCNE-CTF levels. KCNE-CTFs are then cleaved by PS/γ-secretases to generate KCNE intracellular domains (KCNE-ICDs). B ) Western blot analysis of human KCNE1 full-length (F.L.) and its CTF expressed in B104 cells. KCNE1-CTF levels were increased by treatment with DAPT and further elevated by cotreatment with PMA, while partially decreased by TAPI-1. C ) Western blot analysis of human KCNE2 F.L. and its CTF expressed in B104 cells. Similar to KCNE1, KCNE2-CTF levels are also increased by DAPT treatment and further elevated by cotreatment with PMA.

    Techniques Used: Western Blot

    6) Product Images from "BACE1 and presenilin/?-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels"

    Article Title: BACE1 and presenilin/?-secretase regulate proteolytic processing of KCNE1 and 2, auxiliary subunits of voltage-gated potassium channels

    Journal: The FASEB Journal

    doi: 10.1096/fj.12-214056

    Putative BACE1 and PS/γ-secretase cleavage sites of human KCNE1 and KCNE2.
    Figure Legend Snippet: Putative BACE1 and PS/γ-secretase cleavage sites of human KCNE1 and KCNE2.

    Techniques Used:

    Elevated BACE1 activity increases KCNE1- and KCNE2-CTF levels in B104 rat neuroblastoma cells. A ) Overexpression of human BACE1 increased KCNE1-CTF levels in B104 cells. DAPT treatment further enhanced KCNE1-CTF levels in control and BACE1-overexpressing cells. B ) KCNE2-CTF levels were also elevated by BACE1 overexpression in B104 cells.
    Figure Legend Snippet: Elevated BACE1 activity increases KCNE1- and KCNE2-CTF levels in B104 rat neuroblastoma cells. A ) Overexpression of human BACE1 increased KCNE1-CTF levels in B104 cells. DAPT treatment further enhanced KCNE1-CTF levels in control and BACE1-overexpressing cells. B ) KCNE2-CTF levels were also elevated by BACE1 overexpression in B104 cells.

    Techniques Used: Activity Assay, Over Expression

    BACE1 and PS/γ-secretase activities regulate endogenous KCNE1-CTF levels. A ) Detection of endogenous KCNE1 full-length (F.L.) and CTF bands in cultured mouse cardiomyocytes. DAPT (5 μM) treatment increased endogenous KCNE1-CTF levels. B ) Endogenous KCNE1-CTF levels were also increased by BACE1 overexpression in mouse cardiomyocytes.
    Figure Legend Snippet: BACE1 and PS/γ-secretase activities regulate endogenous KCNE1-CTF levels. A ) Detection of endogenous KCNE1 full-length (F.L.) and CTF bands in cultured mouse cardiomyocytes. DAPT (5 μM) treatment increased endogenous KCNE1-CTF levels. B ) Endogenous KCNE1-CTF levels were also increased by BACE1 overexpression in mouse cardiomyocytes.

    Techniques Used: Cell Culture, Over Expression

    PS/γ-secretase activity regulates generation of KCNE1- and KCNE2-ICDs. A ) Western blot analysis showed that KCNE1-ICD is specifically regulated by PS/γ-secretase activity in B104 cells stably expressing full-length KCNE1. Epoxomicin, a proteasome inhibitor, prevented KCNE1-ICD degradation. B ) KCNE2-ICD is detected in the similar conditions.
    Figure Legend Snippet: PS/γ-secretase activity regulates generation of KCNE1- and KCNE2-ICDs. A ) Western blot analysis showed that KCNE1-ICD is specifically regulated by PS/γ-secretase activity in B104 cells stably expressing full-length KCNE1. Epoxomicin, a proteasome inhibitor, prevented KCNE1-ICD degradation. B ) KCNE2-ICD is detected in the similar conditions.

    Techniques Used: Activity Assay, Western Blot, Stable Transfection, Expressing

    KCNE1 and KCNE2 undergo sequential cleavage mediated by α- and PS/γ-secretases. A ) Schematic diagram showing sequential cleavage of KCNE1 and KCNE2 by BACE1, α-, and PS/γ-secretases. α-Secretase inhibitor (TAPI-1) or BACE1 inhibitor ( D R9) decreases generation of KCNE C-terminal fragments (KCNE-CTFs), while PMA, an α-secretase activator, increases KCNE-CTF levels. KCNE-CTFs are then cleaved by PS/γ-secretases to generate KCNE intracellular domains (KCNE-ICDs). B ) Western blot analysis of human KCNE1 full-length (F.L.) and its CTF expressed in B104 cells. KCNE1-CTF levels were increased by treatment with DAPT and further elevated by cotreatment with PMA, while partially decreased by TAPI-1. C ) Western blot analysis of human KCNE2 F.L. and its CTF expressed in B104 cells. Similar to KCNE1, KCNE2-CTF levels are also increased by DAPT treatment and further elevated by cotreatment with PMA.
    Figure Legend Snippet: KCNE1 and KCNE2 undergo sequential cleavage mediated by α- and PS/γ-secretases. A ) Schematic diagram showing sequential cleavage of KCNE1 and KCNE2 by BACE1, α-, and PS/γ-secretases. α-Secretase inhibitor (TAPI-1) or BACE1 inhibitor ( D R9) decreases generation of KCNE C-terminal fragments (KCNE-CTFs), while PMA, an α-secretase activator, increases KCNE-CTF levels. KCNE-CTFs are then cleaved by PS/γ-secretases to generate KCNE intracellular domains (KCNE-ICDs). B ) Western blot analysis of human KCNE1 full-length (F.L.) and its CTF expressed in B104 cells. KCNE1-CTF levels were increased by treatment with DAPT and further elevated by cotreatment with PMA, while partially decreased by TAPI-1. C ) Western blot analysis of human KCNE2 F.L. and its CTF expressed in B104 cells. Similar to KCNE1, KCNE2-CTF levels are also increased by DAPT treatment and further elevated by cotreatment with PMA.

    Techniques Used: Western Blot

    7) Product Images from "Aldosterone downregulates delayed rectifier potassium currents through an angiotensin type 1 receptor-dependent mechanism"

    Article Title: Aldosterone downregulates delayed rectifier potassium currents through an angiotensin type 1 receptor-dependent mechanism

    Journal: American Journal of Translational Research

    doi:

    Effects of enalapril (ENA) or losartan (LOS) on the decreased protein expression of I Ks subunits KCNQ1 and KCNE1 by aldosterone (ALD). Cells were incubated with aldosterone (1 μmol·L -1 ) alone or accompanied with enalapril (1 μmol·L -1 ), losartan (1 μmol·L -1 ) for 24 h. We used western blot to assess I Ks subunits KCNQ1 and KCNE1. A. Representative immunoblots for KCNQ1, KCNE1 proteins along with internal standard GAPDH. B. Mean ± SEM expression levels presented as fold change compared to the control group’s mean value and the quantification of the band intensities were normalized with GAPDH ( n = 3). ** P
    Figure Legend Snippet: Effects of enalapril (ENA) or losartan (LOS) on the decreased protein expression of I Ks subunits KCNQ1 and KCNE1 by aldosterone (ALD). Cells were incubated with aldosterone (1 μmol·L -1 ) alone or accompanied with enalapril (1 μmol·L -1 ), losartan (1 μmol·L -1 ) for 24 h. We used western blot to assess I Ks subunits KCNQ1 and KCNE1. A. Representative immunoblots for KCNQ1, KCNE1 proteins along with internal standard GAPDH. B. Mean ± SEM expression levels presented as fold change compared to the control group’s mean value and the quantification of the band intensities were normalized with GAPDH ( n = 3). ** P

    Techniques Used: Expressing, Incubation, Western Blot

    8) Product Images from "Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model"

    Article Title: Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

    Journal: BMC Medicine

    doi: 10.1186/1741-7015-2-30

    Protein localization of KCNQ1, KCNE1, SLC12A2 and GJB2 in the cochlear lateral wall of Slc26a4 +/+ and Slc26a4 -/- mice. a-f: bars: 10 μm. SMC, strial marginal cells; SV, stria vascularis; BC, basal cells; SL, spiral ligament.
    Figure Legend Snippet: Protein localization of KCNQ1, KCNE1, SLC12A2 and GJB2 in the cochlear lateral wall of Slc26a4 +/+ and Slc26a4 -/- mice. a-f: bars: 10 μm. SMC, strial marginal cells; SV, stria vascularis; BC, basal cells; SL, spiral ligament.

    Techniques Used: Mouse Assay

    9) Product Images from "Doxorubicin induces caspase-mediated proteolysis of KV7.1"

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0162-z

    Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM
    Figure Legend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM

    Techniques Used: Expressing, Western Blot, Immunoprecipitation, Binding Assay, Construct, Transfection, Mutagenesis, Negative Control

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    Alomone Labs anti kcne1 isk antibody
    Cleavage of K v 7.1 in physiology and pathophysiology. A , Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both co-expressed with <t>KCNE1.</t> B , Mean currents amplitude was plotted versus voltage to obtain current-voltage (I-V) relationships in cells expressing K v 7.1-MYC or K v 7.1-D459A-MYC and KCNE1 treated with 500 nmol/L staurosporine for 10 – 12 hours. Statistics were tested with Two-Way ANOVA followed by Bonferroni posttests. C , Western blot analysis of cells used for patch-clamp recordings. D , Schematic illustration to highlight the position of G460 and calmodulin binding site in helix A (upper left panel). Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A transfected cells served as negative controls (lower left panel). Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with One-Way-ANOVA followed by Bonferroni’s Multiple Comparison test. (right panel). (C) anti-K v 7.1 antibody, anti-KCNE1 antibody. (D) anti-MYC antibody, anti-β-actin antibody.
    Anti Kcne1 Isk Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cleavage of K v 7.1 in physiology and pathophysiology. A , Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both co-expressed with KCNE1. B , Mean currents amplitude was plotted versus voltage to obtain current-voltage (I-V) relationships in cells expressing K v 7.1-MYC or K v 7.1-D459A-MYC and KCNE1 treated with 500 nmol/L staurosporine for 10 – 12 hours. Statistics were tested with Two-Way ANOVA followed by Bonferroni posttests. C , Western blot analysis of cells used for patch-clamp recordings. D , Schematic illustration to highlight the position of G460 and calmodulin binding site in helix A (upper left panel). Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A transfected cells served as negative controls (lower left panel). Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with One-Way-ANOVA followed by Bonferroni’s Multiple Comparison test. (right panel). (C) anti-K v 7.1 antibody, anti-KCNE1 antibody. (D) anti-MYC antibody, anti-β-actin antibody.

    Journal: bioRxiv

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    doi: 10.1101/259242

    Figure Lengend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. A , Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both co-expressed with KCNE1. B , Mean currents amplitude was plotted versus voltage to obtain current-voltage (I-V) relationships in cells expressing K v 7.1-MYC or K v 7.1-D459A-MYC and KCNE1 treated with 500 nmol/L staurosporine for 10 – 12 hours. Statistics were tested with Two-Way ANOVA followed by Bonferroni posttests. C , Western blot analysis of cells used for patch-clamp recordings. D , Schematic illustration to highlight the position of G460 and calmodulin binding site in helix A (upper left panel). Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A transfected cells served as negative controls (lower left panel). Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with One-Way-ANOVA followed by Bonferroni’s Multiple Comparison test. (right panel). (C) anti-K v 7.1 antibody, anti-KCNE1 antibody. (D) anti-MYC antibody, anti-β-actin antibody.

    Article Snippet: Antibodies The following antibodies were used: rabbit anti-β-actin (A2066, Sigma-Aldrich, St. Louis, USA), rabbit anti-caspase 3 (8G10, Cell Signaling, Cambridge, UK), rabbit anti-calmodulin (ab45689, Abcam, Cambridge, UK), mouse anti-GAPDH (MAB374, Millipore, Billerica, USA), rabbit anti-KCNE1 (APC-163, Alomone Labs, Jerusalem, Israel), rabbit anti-Kv 7.1 (ab77701, Abcam, Cambridge, UK), mouse anti-MYC (9B11, Cell Signaling, Cambridge, UK), goat anti-MYC (GTX29106, GeneTex Inc., Irvine, USA).

    Techniques: Expressing, Western Blot, Patch Clamp, Binding Assay, Construct, Transfection

    Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM

    Journal: Communications Biology

    Article Title: Doxorubicin induces caspase-mediated proteolysis of KV7.1

    doi: 10.1038/s42003-018-0162-z

    Figure Lengend Snippet: Cleavage of K v 7.1 in physiology and pathophysiology. a Representative current traces for K v 7.1-MYC and K v 7.1-D459A-MYC, both coexpressed with KCNE1. b Mean currents amplitude was plotted versus voltage to obtain current−voltage (I−V) relationships in cells expressing K v 7.1-MYC ( n = 39 for vehicle, n = 16 for staurosporine treatment) or K v 7.1-D459A-MYC ( n = 27 for vehicle, n = 17 for staurosporine treatment) and KCNE1 treated with 500 nmol per L staurosporine for 10–12 h. Statistics were tested with two-way ANOVA followed by Bonferroni post-tests. c Immunoblot analysis of HeLa cells coexpressing Kv7.1 with KCNE1-MYC treated with 1 µM staurosporine for 4.5 h. Untransfected (Ø) and vehicle-treated cells served as negative controls. d Biotinylating study analyzed by immunoblots of Hek 293 cells coexpressing K v 7.1 and KCNE1-MYC treated with 1 µM staurosporine for 3 h. Untransfected (Ø) cells as well as cells not treated with biotin served as negative controls. IP Immunoprecipitation. TL total lysate. e Schematic illustration to highlight the position of G460 and A372 and calmodulin binding site in helix A. Western blot analysis of HeLa cell lysates overexpressing indicated constructs. Untransfected (Ø) and K v 7.1-D459A-transfected cells served as negative controls. Densitometric analysis of four independent experiments of CTF2 band intensity normalized to K v 7.1 full-length band intensity. Statistics were tested with one-way-ANOVA followed by Bonferroni’s Multiple Comparison test. f Coimmunoprecipitation study analyzed by immunoblots of HeLa cells overexpressing wild-type K v 7.1 and the A372D mutant with endogenous calmodulin. IP Immunoprecipitation with anti-K v 7.1 antibody, IB Immunoblot, TL total lysate. Untransfected cells (Ø) served as negative control. c Anti-KCNE1 antibody, anti-caspase 3 antibody. c , d Anti-K v 7.1 antibody, anti-GAPDH antibody. e Anti-β-actin antibody. e , f Anti-MYC antibody. f Anti-calmodulin antibody. All graphs are shown as mean and error bars as SEM

    Article Snippet: The following antibodies were used: rabbit anti-β-actin (A2066, Sigma-Aldrich, St. Louis, USA), rabbit anti-caspase 3 (8G10, Cell Signaling, Cambridge, UK), rabbit anti-calmodulin (ab45689, Abcam, Cambridge, UK), rabbit anti-Eef2 (eukaryotic translation elongation factor 2, ab33523, Abcam, Cambridge, UK), mouse anti-GAPDH (MAB374, Millipore, Billerica, USA), rabbit anti-KCNE1 (APC-163, Alomone Labs, Jerusalem, Israel), rabbit anti-Kv7.1 (ab77701, Abcam, Cambridge, UK), mouse anti-MYC (9B11, Cell Signaling, Cambridge, UK), goat anti-MYC (GTX29106, GeneTex Inc., Irvine, USA).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Binding Assay, Construct, Transfection, Mutagenesis, Negative Control

    Inhibition of protein synthesis does not prevent prKCNE1 assembly with KCNQ1. (A) Whole-cell currents recorded at +50mV from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as a percentage of the current recorded prior to CHX (50 μg/ml) or EtOH treatment (N ≥ 5 for all time points). Arrow indicates when CHX or EtOH was added. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or CHX (bottom traces) for 6h, and then injected with prKCNE1 and further incubated in the presence of CHX or EtOH for an additional 8h. (C) Average currents recorded at +50mV (left panel) and average V ½ values (right panel) obtained from the EtOH-treated (open bar, N = 9) or CHX-treated (solid bar, N = 11) oocytes illustrated in Fig. 2B.

    Journal: Channels (Austin, Tex.)

    Article Title: KCNQ1/KCNE1 ASSEMBLY, CO-TRANSLATION NOT REQUIRED

    doi:

    Figure Lengend Snippet: Inhibition of protein synthesis does not prevent prKCNE1 assembly with KCNQ1. (A) Whole-cell currents recorded at +50mV from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as a percentage of the current recorded prior to CHX (50 μg/ml) or EtOH treatment (N ≥ 5 for all time points). Arrow indicates when CHX or EtOH was added. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or CHX (bottom traces) for 6h, and then injected with prKCNE1 and further incubated in the presence of CHX or EtOH for an additional 8h. (C) Average currents recorded at +50mV (left panel) and average V ½ values (right panel) obtained from the EtOH-treated (open bar, N = 9) or CHX-treated (solid bar, N = 11) oocytes illustrated in Fig. 2B.

    Article Snippet: Oocyte membranes were prepared using a previously published method and western blotting was performed as described previously., Immunoblots for KCNE1 proteins were performed overnight at 4°C with anti-KCNE1 (1:200; Alomone Labs, Jerusalem, Israel), and for KCNQ1 proteins for 1h at room temperature with a goat anti-KCNQ1 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, USA).

    Techniques: Inhibition, Injection, Expressing, Incubation

    Time-course for prKCNE1 modulation of KCNQ1 channels. (A) Schematic showing injection/incubation protocol. (B) Representative current traces recorded from KCNQ1-expressing oocytes at 2, 4, 6 and 18h following prKCNE1 injection. (C) V ½ values for whole-cell currents recorded from oocytes injected with KCNQ1 cRNA (open bar) and KCNQ1 cRNA and prKCNE1 (solid bars). The dashed gray line denotes the V ½ obtained for KCNQ1-KCNE1 channels translated by the oocytes. (D) Time-course for prKCNE1 functional interaction with KCNQ1. In this panel, the dashed line indicates ΔV ½ between KCNQ1-only and KCNQ1-KCNE1 channels translated by the oocytes. N: KCNQ1 cRNA = 9; KCNQ1 + KCNE1 cRNA = 13; KCNQ1 + prKCNE1, 2h = 3; 4h = 5; 6h = 12; 18h = 11; 24h = 8.

    Journal: Channels (Austin, Tex.)

    Article Title: KCNQ1/KCNE1 ASSEMBLY, CO-TRANSLATION NOT REQUIRED

    doi:

    Figure Lengend Snippet: Time-course for prKCNE1 modulation of KCNQ1 channels. (A) Schematic showing injection/incubation protocol. (B) Representative current traces recorded from KCNQ1-expressing oocytes at 2, 4, 6 and 18h following prKCNE1 injection. (C) V ½ values for whole-cell currents recorded from oocytes injected with KCNQ1 cRNA (open bar) and KCNQ1 cRNA and prKCNE1 (solid bars). The dashed gray line denotes the V ½ obtained for KCNQ1-KCNE1 channels translated by the oocytes. (D) Time-course for prKCNE1 functional interaction with KCNQ1. In this panel, the dashed line indicates ΔV ½ between KCNQ1-only and KCNQ1-KCNE1 channels translated by the oocytes. N: KCNQ1 cRNA = 9; KCNQ1 + KCNE1 cRNA = 13; KCNQ1 + prKCNE1, 2h = 3; 4h = 5; 6h = 12; 18h = 11; 24h = 8.

    Article Snippet: Oocyte membranes were prepared using a previously published method and western blotting was performed as described previously., Immunoblots for KCNE1 proteins were performed overnight at 4°C with anti-KCNE1 (1:200; Alomone Labs, Jerusalem, Israel), and for KCNQ1 proteins for 1h at room temperature with a goat anti-KCNQ1 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, USA).

    Techniques: Injection, Incubation, Expressing, Functional Assay

    Modulation of KCNQ1 by prKCNE1 requires vesicular trafficking. (A) Whole-cell currents recorded at +50mV following BFA (5 μM) or EtOH treatment from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as percentage of the current recorded prior to treatment (N ≥ 5 for all time points. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or BFA (bottom traces) for 3h, then injected with prKCNE1 and further incubated for 6 or 15h. (C) V ½ calculated from the normalized current data recorded after 6 and 15h incubations. The dashed line in the bottom panel is the V ½ calculated for KCNQ1-only channels and the open symbols are the V ½ values for currents recorded from KCNQ1-expressing oocytes 6 and 15h after prKCNE1 injection under control conditions. N 6 for both conditions at each time point.

    Journal: Channels (Austin, Tex.)

    Article Title: KCNQ1/KCNE1 ASSEMBLY, CO-TRANSLATION NOT REQUIRED

    doi:

    Figure Lengend Snippet: Modulation of KCNQ1 by prKCNE1 requires vesicular trafficking. (A) Whole-cell currents recorded at +50mV following BFA (5 μM) or EtOH treatment from oocytes injected with KCNQ1 and KCNE1 cRNAs expressed as percentage of the current recorded prior to treatment (N ≥ 5 for all time points. (B) Representative whole-cell currents recorded from KCNQ1-expressing oocytes exposed to either EtOH (top traces) or BFA (bottom traces) for 3h, then injected with prKCNE1 and further incubated for 6 or 15h. (C) V ½ calculated from the normalized current data recorded after 6 and 15h incubations. The dashed line in the bottom panel is the V ½ calculated for KCNQ1-only channels and the open symbols are the V ½ values for currents recorded from KCNQ1-expressing oocytes 6 and 15h after prKCNE1 injection under control conditions. N 6 for both conditions at each time point.

    Article Snippet: Oocyte membranes were prepared using a previously published method and western blotting was performed as described previously., Immunoblots for KCNE1 proteins were performed overnight at 4°C with anti-KCNE1 (1:200; Alomone Labs, Jerusalem, Israel), and for KCNQ1 proteins for 1h at room temperature with a goat anti-KCNQ1 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, USA).

    Techniques: Injection, Expressing, Incubation

    Intracellular processing of prKCNE1. (A) Average whole-cell currents from oocytes expressing KCNQ1 alone (N = 8) and those injected with prKCNE1 (N = 7) or prKCNE1-L51H (N = 10). (B) Average current-voltage relationships from oocytes injected with water (▽, N = 8), KCNQ1 cRNA (○, N = 8) or KCNQ1 cRNA + prKCNE1-L51H (◆, N = 10). Injection of KCNE1-L51H reduced KCNQ1 currents by ~40% when compared to LMPG injection. * = P

    Journal: Channels (Austin, Tex.)

    Article Title: KCNQ1/KCNE1 ASSEMBLY, CO-TRANSLATION NOT REQUIRED

    doi:

    Figure Lengend Snippet: Intracellular processing of prKCNE1. (A) Average whole-cell currents from oocytes expressing KCNQ1 alone (N = 8) and those injected with prKCNE1 (N = 7) or prKCNE1-L51H (N = 10). (B) Average current-voltage relationships from oocytes injected with water (▽, N = 8), KCNQ1 cRNA (○, N = 8) or KCNQ1 cRNA + prKCNE1-L51H (◆, N = 10). Injection of KCNE1-L51H reduced KCNQ1 currents by ~40% when compared to LMPG injection. * = P

    Article Snippet: Oocyte membranes were prepared using a previously published method and western blotting was performed as described previously., Immunoblots for KCNE1 proteins were performed overnight at 4°C with anti-KCNE1 (1:200; Alomone Labs, Jerusalem, Israel), and for KCNQ1 proteins for 1h at room temperature with a goat anti-KCNQ1 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, USA).

    Techniques: Expressing, Injection

    P127T LQT5 mutation and Δ109–129 deletion at the KCNE1 distal C-terminus suppress the cAMP-mediated upregulation of I KS current. Representative current traces recorded from CHO cells co-expressing WT KCNQ1+WT yotiao and WT KCNE1 (A), KCNE1 P127T (C), KCNE1 Δ109–129 (E) and KCNE1 V109I (G). Cells were held at −90 mV and stepped to +30 mV for 3 s and then repolarized to −40 mV for 1.5 s. Cells were recorded in the absence (black traces) or presence (red traces) of 200 µM cAMP+0.2 µM okadaic acid. Current–voltage relationships of WT KCNQ1+WT yotiao and WT KCNE1 (B) or KCNE1 P127T (D) or KCNE1 Δ109–129 (F) or KCNE1 V109I (H). Voltage was stepped for 3 s from −50 mV to +60 mV in 10 mV increments followed by repolarization to −40 mV for 1.5 s. Red and black curves represent recordings with or without cAMP+okadaic acid in the patch pipette, respectively. ( n = 6–7). Above +30 mV, the WT I KS and, above +20 mV, the KCNE1 V109I current upregulation induced by cAMP+okadaic acid were significant. * P

    Journal: Journal of Cell Science

    Article Title: Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2

    doi: 10.1242/jcs.147033

    Figure Lengend Snippet: P127T LQT5 mutation and Δ109–129 deletion at the KCNE1 distal C-terminus suppress the cAMP-mediated upregulation of I KS current. Representative current traces recorded from CHO cells co-expressing WT KCNQ1+WT yotiao and WT KCNE1 (A), KCNE1 P127T (C), KCNE1 Δ109–129 (E) and KCNE1 V109I (G). Cells were held at −90 mV and stepped to +30 mV for 3 s and then repolarized to −40 mV for 1.5 s. Cells were recorded in the absence (black traces) or presence (red traces) of 200 µM cAMP+0.2 µM okadaic acid. Current–voltage relationships of WT KCNQ1+WT yotiao and WT KCNE1 (B) or KCNE1 P127T (D) or KCNE1 Δ109–129 (F) or KCNE1 V109I (H). Voltage was stepped for 3 s from −50 mV to +60 mV in 10 mV increments followed by repolarization to −40 mV for 1.5 s. Red and black curves represent recordings with or without cAMP+okadaic acid in the patch pipette, respectively. ( n = 6–7). Above +30 mV, the WT I KS and, above +20 mV, the KCNE1 V109I current upregulation induced by cAMP+okadaic acid were significant. * P

    Article Snippet: Primary antibodies were anti-KCNQ1 (1:1000, Alomone), anti-KCNE1 (1:200, Alomone Labs), anti-yotiao (1:1000, Carmen Dessauer, University of Texas Health Science Center, Houston) and anti-KCNQ1 phospho-S27 (1:250, Robert S. Kass, University of Columbia, New York).

    Techniques: Mutagenesis, Expressing, Transferring

    Effects of KCNE1 C-terminus deletions and LQT5 mutations on I KS currents. (A) A cartoon of KCNE1, indicating the location of the LQT5 mutations and the deletions in the C-terminus. Representative current traces of WT KCNQ1, co-expressed in CHO cells with WT KCNE1 (B), LQT5 mutant V109I (C), LQT5 mutant P127T (D) or with KCNE1 deletion mutants at the distal C-terminus Δ109–129 (E) or the proximal C-terminus Δ69–77 (F). Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −50 mV to +60 mV (or to +100 mV for Δ69–77) in 10 mV increments and then repolarized for 1.5 s to −60 mV. Current–voltage (G) and conductance–voltage (H) relationships of WT KCNQ1+WT KCNE1 or LQT5 mutants ( n = 11–17). Current–voltage (I) and conductance–voltage (J) relationships of WT KCNQ1+WT KCNE1 or deletion mutants ( n = 7–10).

    Journal: Journal of Cell Science

    Article Title: Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2

    doi: 10.1242/jcs.147033

    Figure Lengend Snippet: Effects of KCNE1 C-terminus deletions and LQT5 mutations on I KS currents. (A) A cartoon of KCNE1, indicating the location of the LQT5 mutations and the deletions in the C-terminus. Representative current traces of WT KCNQ1, co-expressed in CHO cells with WT KCNE1 (B), LQT5 mutant V109I (C), LQT5 mutant P127T (D) or with KCNE1 deletion mutants at the distal C-terminus Δ109–129 (E) or the proximal C-terminus Δ69–77 (F). Cells were held at −90 mV. Membrane voltage was stepped for 3 s from −50 mV to +60 mV (or to +100 mV for Δ69–77) in 10 mV increments and then repolarized for 1.5 s to −60 mV. Current–voltage (G) and conductance–voltage (H) relationships of WT KCNQ1+WT KCNE1 or LQT5 mutants ( n = 11–17). Current–voltage (I) and conductance–voltage (J) relationships of WT KCNQ1+WT KCNE1 or deletion mutants ( n = 7–10).

    Article Snippet: Primary antibodies were anti-KCNQ1 (1:1000, Alomone), anti-KCNE1 (1:200, Alomone Labs), anti-yotiao (1:1000, Carmen Dessauer, University of Texas Health Science Center, Houston) and anti-KCNQ1 phospho-S27 (1:250, Robert S. Kass, University of Columbia, New York).

    Techniques: Mutagenesis

    LQT1 and LQT5 mutations disrupt the interaction between KCNQ1 helix C and KCNE1 C-terminus. (A) Representative immunoblot of GST pulldown (GST PD) (upper row) of the His-MBP-tagged KCNE1 C-terminus (residues 67–129, E1CT in diagram) or control His-MBP peptide, by GST-tagged KCNQ1 helix C (residues 535–572, Q1 Helix C, in diagram), illustrating the direct interaction of KCNE1 C-terminus with KCNQ1 helix C ( n = 4). Equal amounts of His-MBP and His-MBP-tagged KCNE1 were used, as seen in the input (lower row). (B) Representative immunoblot of GST pulldown (upper row) of the His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504, Q1CT in diagram) by GST-tagged KCNE1 C-terminus deletion mutants (E1CT in diagram). Inputs are shown in the lower row. (C) Quantification of the pulldowns normalized to input ( n = 5). * P

    Journal: Journal of Cell Science

    Article Title: Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2

    doi: 10.1242/jcs.147033

    Figure Lengend Snippet: LQT1 and LQT5 mutations disrupt the interaction between KCNQ1 helix C and KCNE1 C-terminus. (A) Representative immunoblot of GST pulldown (GST PD) (upper row) of the His-MBP-tagged KCNE1 C-terminus (residues 67–129, E1CT in diagram) or control His-MBP peptide, by GST-tagged KCNQ1 helix C (residues 535–572, Q1 Helix C, in diagram), illustrating the direct interaction of KCNE1 C-terminus with KCNQ1 helix C ( n = 4). Equal amounts of His-MBP and His-MBP-tagged KCNE1 were used, as seen in the input (lower row). (B) Representative immunoblot of GST pulldown (upper row) of the His-tagged WT KCNQ1 C-terminus (residues 352–622 Δ396–504, Q1CT in diagram) by GST-tagged KCNE1 C-terminus deletion mutants (E1CT in diagram). Inputs are shown in the lower row. (C) Quantification of the pulldowns normalized to input ( n = 5). * P

    Article Snippet: Primary antibodies were anti-KCNQ1 (1:1000, Alomone), anti-KCNE1 (1:200, Alomone Labs), anti-yotiao (1:1000, Carmen Dessauer, University of Texas Health Science Center, Houston) and anti-KCNQ1 phospho-S27 (1:250, Robert S. Kass, University of Columbia, New York).

    Techniques:

    LQT5 P127T mutant and deletion mutant Δ109–129 impair phosphorylation of KCNQ1 at S27 during cAMP-dependent stimulation. (A) Representative immunoblots (IB) of HEK 293 cells lysates co-expressing yotiao, WT KCNQ1 and WT, mutated (V109I and P127T) or truncated (Δ109–129) KCNE1 from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid and immunoblots of HEK 293 cells lysates co-expressing yotiao WT KCNE1 and WT KCNQ1 or LQT1 mutant K557E from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid. Blots were probed with antibodies against phosphorylated KCNQ1 S27 (first row from top), KCNQ1 (second row from top), yotiao (third row from top) and KCNE1 (fourth row from top). (B) Quantification of phosphorylated KCNQ1 S27 was calculated by dividing phosphorylated signal to KCNQ1 input (anti-KCNQ1 blot) for both unstimulated and cAMP-stimulated cells (250 µM 8CPT+0.2 µM okadaic acid) and normalized to stimulated WT KCNQ1+WT KCNE1+yotiao ( n = 3–5). * P

    Journal: Journal of Cell Science

    Article Title: Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2

    doi: 10.1242/jcs.147033

    Figure Lengend Snippet: LQT5 P127T mutant and deletion mutant Δ109–129 impair phosphorylation of KCNQ1 at S27 during cAMP-dependent stimulation. (A) Representative immunoblots (IB) of HEK 293 cells lysates co-expressing yotiao, WT KCNQ1 and WT, mutated (V109I and P127T) or truncated (Δ109–129) KCNE1 from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid and immunoblots of HEK 293 cells lysates co-expressing yotiao WT KCNE1 and WT KCNQ1 or LQT1 mutant K557E from cells treated in the absence and presence of 250 µM 8CPT+0.2 µM okadaic acid. Blots were probed with antibodies against phosphorylated KCNQ1 S27 (first row from top), KCNQ1 (second row from top), yotiao (third row from top) and KCNE1 (fourth row from top). (B) Quantification of phosphorylated KCNQ1 S27 was calculated by dividing phosphorylated signal to KCNQ1 input (anti-KCNQ1 blot) for both unstimulated and cAMP-stimulated cells (250 µM 8CPT+0.2 µM okadaic acid) and normalized to stimulated WT KCNQ1+WT KCNE1+yotiao ( n = 3–5). * P

    Article Snippet: Primary antibodies were anti-KCNQ1 (1:1000, Alomone), anti-KCNE1 (1:200, Alomone Labs), anti-yotiao (1:1000, Carmen Dessauer, University of Texas Health Science Center, Houston) and anti-KCNQ1 phospho-S27 (1:250, Robert S. Kass, University of Columbia, New York).

    Techniques: Mutagenesis, Western Blot, Expressing

    LQT1 and LQT5 mutations do not affect the channel trafficking to the plasma membrane. (A) TIRF fluorescence images of CHO cells co-expressing CFP-tagged WT KCNQ1 and GPI-citrine. (B) Epi-fluorescence (left panels) and TIRF fluorescence (right panels) images of CHO cells expressing CFP-tagged WT KCNQ1+KCNE1 (upper panels) or CFP-tagged G589D LQT1 mutant+KCNE1 (lower panels). (C) TIRF fluorescence images of CHO cells co-expressing CFP-tagged helix C LQT1 mutants with KCNE1. (D) YFP-tagged WT KCNE1 or LQT5 mutant co-expressed with WT KCNQ1. Quantification of the TIRF fluorescent signals of CFP-tagged WT KCNQ1 and LQT1 mutants (E) and of YFP-tagged WT and LQT5 mutant (F) as normalized to membrane signal area ( n = 16–60). *** P

    Journal: Journal of Cell Science

    Article Title: Long QT mutations at the interface between KCNQ1 helix C and KCNE1 disrupt IKS regulation by PKA and PIP2

    doi: 10.1242/jcs.147033

    Figure Lengend Snippet: LQT1 and LQT5 mutations do not affect the channel trafficking to the plasma membrane. (A) TIRF fluorescence images of CHO cells co-expressing CFP-tagged WT KCNQ1 and GPI-citrine. (B) Epi-fluorescence (left panels) and TIRF fluorescence (right panels) images of CHO cells expressing CFP-tagged WT KCNQ1+KCNE1 (upper panels) or CFP-tagged G589D LQT1 mutant+KCNE1 (lower panels). (C) TIRF fluorescence images of CHO cells co-expressing CFP-tagged helix C LQT1 mutants with KCNE1. (D) YFP-tagged WT KCNE1 or LQT5 mutant co-expressed with WT KCNQ1. Quantification of the TIRF fluorescent signals of CFP-tagged WT KCNQ1 and LQT1 mutants (E) and of YFP-tagged WT and LQT5 mutant (F) as normalized to membrane signal area ( n = 16–60). *** P

    Article Snippet: Primary antibodies were anti-KCNQ1 (1:1000, Alomone), anti-KCNE1 (1:200, Alomone Labs), anti-yotiao (1:1000, Carmen Dessauer, University of Texas Health Science Center, Houston) and anti-KCNQ1 phospho-S27 (1:250, Robert S. Kass, University of Columbia, New York).

    Techniques: Fluorescence, Expressing, Mutagenesis