apc064  (Alomone Labs)


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    Alomone Labs apc064
    Apc064, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    apc064 - by Bioz Stars, 2022-05
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    Alomone Labs anti kca3 1
    Complementary roles of ECM, β1-integrin, <t>KCa3.1</t> and TRPC4 channels during alveolar repair process. The presence of fibronectin matrix evokes an increase in membrane expression of β1-integrin, KCa3.1 and TRPC4, an elevation of steady-state [Ca 2+ ] i and a stimulation of ATII wound healing. This stimulation is dependent, at least in part on KCa3.1 and TRPC4 channel activity. Our data also highlighted a relationship between KCa3.1 and β1-integrin
    Anti Kca3 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti kca3 1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti kca3 1 - by Bioz Stars, 2022-05
    93/100 stars
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    Complementary roles of ECM, β1-integrin, KCa3.1 and TRPC4 channels during alveolar repair process. The presence of fibronectin matrix evokes an increase in membrane expression of β1-integrin, KCa3.1 and TRPC4, an elevation of steady-state [Ca 2+ ] i and a stimulation of ATII wound healing. This stimulation is dependent, at least in part on KCa3.1 and TRPC4 channel activity. Our data also highlighted a relationship between KCa3.1 and β1-integrin

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Complementary roles of ECM, β1-integrin, KCa3.1 and TRPC4 channels during alveolar repair process. The presence of fibronectin matrix evokes an increase in membrane expression of β1-integrin, KCa3.1 and TRPC4, an elevation of steady-state [Ca 2+ ] i and a stimulation of ATII wound healing. This stimulation is dependent, at least in part on KCa3.1 and TRPC4 channel activity. Our data also highlighted a relationship between KCa3.1 and β1-integrin

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques: Expressing, Activity Assay

    Expression of TRPC channels in ATII cells and complementary roles of TRPC4 and KCa3.1 in wound repair on fibronectin matrix. Representative agarose gel showing PCR products amplified from ATII cDNA with PCR primer pairs designed from rat TRPC channels ( a ). TRPC channel relative mRNA expressions were normalized to β-actin and compared between no coating and fibronectin matrix conditions ( b , n = 16–20). c . Levels of TRPC4 protein expression in membrane fractions were measured by immunoblotting using specific anti-TRPC4 channel antibody and band intensities were compared between control (no coating) and fibronectin matrix conditions ( c , right panel, n = 11). A representative immunoblot is shown in the left panel. d . ATII cells, cultured in the absence (−) or presence (+) of a fibronectin coating, were injured mechanically and the wound-healing rates (μm 2 /h) over a 24-h period were then compared in control condition (−), in presence of the TRPC4 inhibitor ML-204 (100 μM, +) (n = 8) or a combination of ML-204 (100 μM) and TRAM-34 (20 μM) ( d , n = 7). * p

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Expression of TRPC channels in ATII cells and complementary roles of TRPC4 and KCa3.1 in wound repair on fibronectin matrix. Representative agarose gel showing PCR products amplified from ATII cDNA with PCR primer pairs designed from rat TRPC channels ( a ). TRPC channel relative mRNA expressions were normalized to β-actin and compared between no coating and fibronectin matrix conditions ( b , n = 16–20). c . Levels of TRPC4 protein expression in membrane fractions were measured by immunoblotting using specific anti-TRPC4 channel antibody and band intensities were compared between control (no coating) and fibronectin matrix conditions ( c , right panel, n = 11). A representative immunoblot is shown in the left panel. d . ATII cells, cultured in the absence (−) or presence (+) of a fibronectin coating, were injured mechanically and the wound-healing rates (μm 2 /h) over a 24-h period were then compared in control condition (−), in presence of the TRPC4 inhibitor ML-204 (100 μM, +) (n = 8) or a combination of ML-204 (100 μM) and TRAM-34 (20 μM) ( d , n = 7). * p

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques: Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Cell Culture

    Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a . Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b . Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c . The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix ( right panel , n = 11). * p

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Cellular co-distribution, co-immunoprecipitation and membrane expression of β1-integrin and KCa3.1 channels. a . Representative immunofluorescence images of KCa3.1 and β1-integrin staining performed on ATII cells using anti-KCa3.1, anti-β1-integrin, anti-rabbit 633 (for KCa3.1 detection) and anti-mouse 488 (for β1-integrin detection) antibodies. Color superposition shows similar cellular distribution of KCa3.1 and β1-integrin in ATII cells (merge panel, Scale bars, 10 μm). No or diffuse signal was detected with the Alexa fluor 488 and Alexa fluor 633 coupled secondary antibodies in control experiments (negative controls). b . Representative immunoblots showing β1-integrin and KCa3.1 co-immunoprecipitations. β1-integrin (upper panels, IB: β1-integrin) and KCa3.1 (lower panels, IB: KCa3.1) proteins were revealed with specific antibodies after β1-integrin and KCa3.1 immunoprecipitation with anti-β1-integrin (lane 2 « β1-integrin IP ») or anti-KCa3.1 (lane 3 « KCa3.1 IP ») antibodies in ATII cell extracts. Endogenous expression of β1-integrin and KCa3.1 proteins in ATII cell lysate is also shown in lane 1, « Total Lysate ». Lanes 4 and 5 are negative control assays showing an absence of band in IB (IB β1-integrin and IB KCa3.1) after IP in the absence of lysate (lane 4, « Negative IP Control (no lysate) ») and in the absence of β-integrin and KCa3.1 antibodies (lane 5, « Negative IP control (no antibody) »). c . The level of β1-integrin and KCa3.1 channel expression in membrane fractions were determined by immunoblotting using anti-β1-integrin and anti-KCa3.1 antibodies. A representative immunoblot is shown in the left panel. The band intensities were compared in control condition (no coating, −) and in the presence of a fibronectin (+) matrix ( right panel , n = 11). * p

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques: Immunoprecipitation, Expressing, Immunofluorescence, Staining, Western Blot, Negative Control

    Impact of KCa3.1 inhibition on fibronectin-stimulated ATII wound repair. ATII cell monolayers, grown in the absence (no coating) or presence of fibronectin coating, were injured mechanically and wound-healing rates were measured over a 24-h period, in the presence or absence of the KCa3.1 inhibitor TRAM-34. Representative photographs (x4 magnification) at time 0 (T 0 ) and 24 h (T 24 ) after injury in each condition are presented in ( a ). The wound edge is indicated by the dotted lines. b . Mean wound-closure rates (μm 2 /h) are compared in control (0) and TRAM-34 (5 and 10 μM) treated monolayers, in the absence or presence of fibronectin coating (n = 6). * p

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Impact of KCa3.1 inhibition on fibronectin-stimulated ATII wound repair. ATII cell monolayers, grown in the absence (no coating) or presence of fibronectin coating, were injured mechanically and wound-healing rates were measured over a 24-h period, in the presence or absence of the KCa3.1 inhibitor TRAM-34. Representative photographs (x4 magnification) at time 0 (T 0 ) and 24 h (T 24 ) after injury in each condition are presented in ( a ). The wound edge is indicated by the dotted lines. b . Mean wound-closure rates (μm 2 /h) are compared in control (0) and TRAM-34 (5 and 10 μM) treated monolayers, in the absence or presence of fibronectin coating (n = 6). * p

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques: Inhibition

    Involvement of KCa3.1 channels in ATII cell proliferation. Subconfluent ATII cells grown for 3 days in the absence ( a ) or presence of fibronectin coating ( b ) were exposed to increasing TRAM-34 concentrations (5, 10 or 20 μM) for 24 h. Cell proliferation was estimated at day 3 (T0, before treatment) and day 4 (T24h) in each condition. (n = 8). * p

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Involvement of KCa3.1 channels in ATII cell proliferation. Subconfluent ATII cells grown for 3 days in the absence ( a ) or presence of fibronectin coating ( b ) were exposed to increasing TRAM-34 concentrations (5, 10 or 20 μM) for 24 h. Cell proliferation was estimated at day 3 (T0, before treatment) and day 4 (T24h) in each condition. (n = 8). * p

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques:

    Delayed ATII wound repair and reduced cell proliferation and migration after KCa3.1 silencing in the presence of fibronectin coating. ATII cells were transfected with negative control siRNA (siRNA control) or siRNAs directed against KCa3.1 (siKCa3.1) and then seeded in the absence or presence of fibronectin coating. KCa3.1 silencing was verified by PCR ( a , n = 7). A representative agarose gel is presented on the left. ( b ) Wound-healing rates (in μm 2 /h) were measured at 24 h after injury among cells transfected with control or KCa3.1 siRNAs in the absence of coating (left) and in the presence of fibronectin matrix (right) (n = 7). The number of ATII cells was counted at day 3 of culture after transfection with control or KCa3.1 siRNAs (n = 14, c ). The number of migrating ATII cells, transfected 3 days before with control or KCa3.1 siRNAs, was measured in a Boyden type chamber over an 18-h period in the absence (no coating) or presence of fibronectin (n = 6, d ). * p

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Delayed ATII wound repair and reduced cell proliferation and migration after KCa3.1 silencing in the presence of fibronectin coating. ATII cells were transfected with negative control siRNA (siRNA control) or siRNAs directed against KCa3.1 (siKCa3.1) and then seeded in the absence or presence of fibronectin coating. KCa3.1 silencing was verified by PCR ( a , n = 7). A representative agarose gel is presented on the left. ( b ) Wound-healing rates (in μm 2 /h) were measured at 24 h after injury among cells transfected with control or KCa3.1 siRNAs in the absence of coating (left) and in the presence of fibronectin matrix (right) (n = 7). The number of ATII cells was counted at day 3 of culture after transfection with control or KCa3.1 siRNAs (n = 14, c ). The number of migrating ATII cells, transfected 3 days before with control or KCa3.1 siRNAs, was measured in a Boyden type chamber over an 18-h period in the absence (no coating) or presence of fibronectin (n = 6, d ). * p

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques: Migration, Transfection, Negative Control, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Impact of KCa3.1 inhibition on ATII cell motility. ATII cell migration was evaluated using Boyden-type assays ( a ) and single cell tracking in live time-lapse videomicroscopy ( b ). The number of migrating ATII cells were measured over an 18-h period in the absence (0) or presence of 5, 10 or 20 μM TRAM-34 and compared in the absence ( a , no fibronectin coating, left panel) or presence of fibronectin ( a , right panel) (5 fields/insert, 3 inserts/condition, n = 4-6). 2D cell migration rates ( b ) were evaluated by ATII single-cell tracking (~15 cells/field, 2 fields/condition/experiment, n = 6) over a 24-h period in the absence (0) or presence of TRAM-34 (5, 10 and 20 μM). Representative trajectories of ATII cells in the absence or presence of a fibronectin matrix, in control condition and in the presence of 20 μM TRAM-34 are presented in the top panel ( b ). Colors indicate 0-h (blue) to 24-h (red) time points. * p

    Journal: Respiratory Research

    Article Title: Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

    doi: 10.1186/s12931-015-0263-x

    Figure Lengend Snippet: Impact of KCa3.1 inhibition on ATII cell motility. ATII cell migration was evaluated using Boyden-type assays ( a ) and single cell tracking in live time-lapse videomicroscopy ( b ). The number of migrating ATII cells were measured over an 18-h period in the absence (0) or presence of 5, 10 or 20 μM TRAM-34 and compared in the absence ( a , no fibronectin coating, left panel) or presence of fibronectin ( a , right panel) (5 fields/insert, 3 inserts/condition, n = 4-6). 2D cell migration rates ( b ) were evaluated by ATII single-cell tracking (~15 cells/field, 2 fields/condition/experiment, n = 6) over a 24-h period in the absence (0) or presence of TRAM-34 (5, 10 and 20 μM). Representative trajectories of ATII cells in the absence or presence of a fibronectin matrix, in control condition and in the presence of 20 μM TRAM-34 are presented in the top panel ( b ). Colors indicate 0-h (blue) to 24-h (red) time points. * p

    Article Snippet: After blocking, the membranes were incubated with anti-KCa3.1 (APC-064, Alomone Labs), anti-β1-integrin (610467, BD Biosciences) or anti-TRPC4 (ACC-018, Alomone Labs) antibodies.

    Techniques: Inhibition, Migration, Single Cell Tracking

    Impact of AMPK activity on KCa3.1currents. Short-circuit currents ( I sc ) measured in Ussing chamber on Nuli cell monolayers cultured for 4 to 6 wk, at air-liquid-interface. A : applying the KCa3.1 potentiator 1-EBIO (1 mM) to the basolateral side of the monolayer caused an increase in I sc , which was inhibited by a basolateral application of the KCa3.1 inhibitor Tram-34 (5 μM). This result confirms the presence of KCa3.1 at the basolateral membrane of Nuli cell monolayers. B : similarly, applying 1-EBIO (1 mM) to the apical side of the monolayer caused an increase in I sc , which was inhibited by apical application of Tram-34 (5 μM), confirming the presence of KCa3.1 at the apical membrane of Nuli cell monolayers. C : basolateral addition of Tram-34 following apical stimulation of KCa3.1 by 1-EBIO did not result in a significant I sc decrease, confirming the side specificity of the action of Tram-34 seen in B. D – F : impact of AMPK activity on KCa3.1 currents was evaluated by I sc measured in nontreated (Ctl), AICAR-treated (1 mM, 1 h, apical and basolateral side), or AICAR + Compound-C treated (10 μM, 1 h, apical and basolateral side) monolayers. AICAR is used to activate AMPK via the internal production of 5-amino-4-imidazole-carboxamide ribotide (ZMP). Basal short-circuit currents ( I basal , D ; n = 11), 1-EBIO-induced currents ( I 1-EBIO , E ; n = 11), and 1-EBIO-induced, Tram-34 inhibited currents ( I Tram-34 , F ; n = 11) were then compared in control, AICAR, and AICAR + Compound-C-treated monolayers. The presence of Compound-C succeeded to reverse the effect of AICAR on the KCa3.1-dependent short-circuit currents. These results argue for a control of the KCa3.1 activity by AMPK in Nuli monolayers.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    doi: 10.1152/ajpcell.00418.2008

    Figure Lengend Snippet: Impact of AMPK activity on KCa3.1currents. Short-circuit currents ( I sc ) measured in Ussing chamber on Nuli cell monolayers cultured for 4 to 6 wk, at air-liquid-interface. A : applying the KCa3.1 potentiator 1-EBIO (1 mM) to the basolateral side of the monolayer caused an increase in I sc , which was inhibited by a basolateral application of the KCa3.1 inhibitor Tram-34 (5 μM). This result confirms the presence of KCa3.1 at the basolateral membrane of Nuli cell monolayers. B : similarly, applying 1-EBIO (1 mM) to the apical side of the monolayer caused an increase in I sc , which was inhibited by apical application of Tram-34 (5 μM), confirming the presence of KCa3.1 at the apical membrane of Nuli cell monolayers. C : basolateral addition of Tram-34 following apical stimulation of KCa3.1 by 1-EBIO did not result in a significant I sc decrease, confirming the side specificity of the action of Tram-34 seen in B. D – F : impact of AMPK activity on KCa3.1 currents was evaluated by I sc measured in nontreated (Ctl), AICAR-treated (1 mM, 1 h, apical and basolateral side), or AICAR + Compound-C treated (10 μM, 1 h, apical and basolateral side) monolayers. AICAR is used to activate AMPK via the internal production of 5-amino-4-imidazole-carboxamide ribotide (ZMP). Basal short-circuit currents ( I basal , D ; n = 11), 1-EBIO-induced currents ( I 1-EBIO , E ; n = 11), and 1-EBIO-induced, Tram-34 inhibited currents ( I Tram-34 , F ; n = 11) were then compared in control, AICAR, and AICAR + Compound-C-treated monolayers. The presence of Compound-C succeeded to reverse the effect of AICAR on the KCa3.1-dependent short-circuit currents. These results argue for a control of the KCa3.1 activity by AMPK in Nuli monolayers.

    Article Snippet: For immunoprecipitation of endogenous KCa3.1 or AMPK-γ1 proteins, precleared soluble lysates were incubated for 1–2 h with a rabbit anti-KCa3.1 antibody (1:100, Alomone Labs, Jerusalem, Israel) or with a rabbit anti-AMPK-γ1 antibody (1:100 or 1:150, Abcam, Cambridge, MA).

    Techniques: Activity Assay, Cell Culture, CTL Assay

    Coimmunoprecipitation of endogenous AMPK-γ1 and KCa3.1 in NuLi cells. Immunoblots showing AMPK-γ1 ( A, B ) and KCa3.1 ( C, D ) proteins from NuLi extracts and AMPK-γ1/KCa3.1 immunoprecipitations. Membranes were blotted with anti-AMPK-γ1 ( A, B ) and anti-KCa3.1 ( C, D ) antibodies, in the presence ( B, D ) or absence ( A, C ) of neutralizing peptide. Endogenous expression of AMPK-γ1 and KCa3.1 proteins in the NuLi cell lysate are presented in lanes 4 of A and C , respectively. Control lanes ( A and C, lanes 1 ) refer to coimmunoprecipitation experiments done in the absence of immunoprecipitating antibody. Control immunoprecipitations were also performed, AMPK-γ1 immunoprecipitation with the AMPK-γ1 antibody ( A, lane 2 ) and KCa3.1 immmunoprecipitation with the KCa3.1 antibody ( C, lane 3 ). Coimmunoprecipitation of endogenous AMPK-γ1 using anti-KCa3.1 antibody is illustrated in A, lane 3 . Conversely, coimmunoprecipitation of endogenous KCa3.1 using anti-AMPK-γ1 antibody is shown at C, lane 2 . Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with AMPK-γ1 and KCa3.1 antibodies, respectively. Because of the higher AMPK-γ1 signal in cell lysates, 1/3 of the total IP was loaded for AMPK-γ1 immunoblot, whereas 2/3 of the total IP was loaded for the KCa3.1 immunoblot. No band was observed when the membranes were blotted with anti-AMPK-γ1 or anti-KCa3.1 antibodies in the presence of their respective neutralizing peptides ( B and D ).

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    doi: 10.1152/ajpcell.00418.2008

    Figure Lengend Snippet: Coimmunoprecipitation of endogenous AMPK-γ1 and KCa3.1 in NuLi cells. Immunoblots showing AMPK-γ1 ( A, B ) and KCa3.1 ( C, D ) proteins from NuLi extracts and AMPK-γ1/KCa3.1 immunoprecipitations. Membranes were blotted with anti-AMPK-γ1 ( A, B ) and anti-KCa3.1 ( C, D ) antibodies, in the presence ( B, D ) or absence ( A, C ) of neutralizing peptide. Endogenous expression of AMPK-γ1 and KCa3.1 proteins in the NuLi cell lysate are presented in lanes 4 of A and C , respectively. Control lanes ( A and C, lanes 1 ) refer to coimmunoprecipitation experiments done in the absence of immunoprecipitating antibody. Control immunoprecipitations were also performed, AMPK-γ1 immunoprecipitation with the AMPK-γ1 antibody ( A, lane 2 ) and KCa3.1 immmunoprecipitation with the KCa3.1 antibody ( C, lane 3 ). Coimmunoprecipitation of endogenous AMPK-γ1 using anti-KCa3.1 antibody is illustrated in A, lane 3 . Conversely, coimmunoprecipitation of endogenous KCa3.1 using anti-AMPK-γ1 antibody is shown at C, lane 2 . Note that the same lysate and IP samples were used in the upper and lower parts of the membranes, blotted with AMPK-γ1 and KCa3.1 antibodies, respectively. Because of the higher AMPK-γ1 signal in cell lysates, 1/3 of the total IP was loaded for AMPK-γ1 immunoblot, whereas 2/3 of the total IP was loaded for the KCa3.1 immunoblot. No band was observed when the membranes were blotted with anti-AMPK-γ1 or anti-KCa3.1 antibodies in the presence of their respective neutralizing peptides ( B and D ).

    Article Snippet: For immunoprecipitation of endogenous KCa3.1 or AMPK-γ1 proteins, precleared soluble lysates were incubated for 1–2 h with a rabbit anti-KCa3.1 antibody (1:100, Alomone Labs, Jerusalem, Israel) or with a rabbit anti-AMPK-γ1 antibody (1:100 or 1:150, Abcam, Cambridge, MA).

    Techniques: Western Blot, Expressing, Immunoprecipitation

    Inside-out experiments illustrating a regulation of the KCa3.1 activity by AMP. Inside-out patch-clamp recording performed on human epithelia kidney HEK-293 cells expressing the Myc-KCa3.1 channel. Recording obtained in 10 μM internal Ca 2+ conditions at an applied membrane potential of −60 mV. Perfusion with a 0 Ca 2+ solution (EGTA) is represented as a filled rectangle. The symbol c refers to the zero current level. A : internal addition of the KCa3.1-specific inhibitor Tram-34 (1 μM) caused a strong current inhibition that was slowly reversible, confirming that the Ca 2+ -sensitive current measured under our experimental conditions corresponds to KCa3.1. Inset , single-channel events, confirming current jumps of 2.4 pA for an unitary conductance of 40 pS as expected for KCa3.1. B : initial addition of 1 mM ATP to the internal medium caused an increase in channel activity to reach a stable current level. Perfusion with a solution containing 1 mM ATP plus 200 μM AMP led to a 46 ± 18% ( n = 17) decrease in channel activity that was reversible following the washout of AMP. C : effect of AMP on channel activity in the absence of ATP. Perfusion with an internal solution containing AMP (200 μM) in the absence of ATP did not affect the time course of the channel rundown process. This result supports an effect of AMP on KCa3.1 that is ATP dependent. D : frequency histogram of the percentage of current inhibition measured at 200 μM AMP obtained from 17 different cells. E : percentage of current inhibition measured as a function of the AMP concentration for ATP at 1 mM. The AMP concentration for half inhibition was estimated at 140 μM. Each data point represents the mean ± SD of at least 3 different experiments.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    doi: 10.1152/ajpcell.00418.2008

    Figure Lengend Snippet: Inside-out experiments illustrating a regulation of the KCa3.1 activity by AMP. Inside-out patch-clamp recording performed on human epithelia kidney HEK-293 cells expressing the Myc-KCa3.1 channel. Recording obtained in 10 μM internal Ca 2+ conditions at an applied membrane potential of −60 mV. Perfusion with a 0 Ca 2+ solution (EGTA) is represented as a filled rectangle. The symbol c refers to the zero current level. A : internal addition of the KCa3.1-specific inhibitor Tram-34 (1 μM) caused a strong current inhibition that was slowly reversible, confirming that the Ca 2+ -sensitive current measured under our experimental conditions corresponds to KCa3.1. Inset , single-channel events, confirming current jumps of 2.4 pA for an unitary conductance of 40 pS as expected for KCa3.1. B : initial addition of 1 mM ATP to the internal medium caused an increase in channel activity to reach a stable current level. Perfusion with a solution containing 1 mM ATP plus 200 μM AMP led to a 46 ± 18% ( n = 17) decrease in channel activity that was reversible following the washout of AMP. C : effect of AMP on channel activity in the absence of ATP. Perfusion with an internal solution containing AMP (200 μM) in the absence of ATP did not affect the time course of the channel rundown process. This result supports an effect of AMP on KCa3.1 that is ATP dependent. D : frequency histogram of the percentage of current inhibition measured at 200 μM AMP obtained from 17 different cells. E : percentage of current inhibition measured as a function of the AMP concentration for ATP at 1 mM. The AMP concentration for half inhibition was estimated at 140 μM. Each data point represents the mean ± SD of at least 3 different experiments.

    Article Snippet: For immunoprecipitation of endogenous KCa3.1 or AMPK-γ1 proteins, precleared soluble lysates were incubated for 1–2 h with a rabbit anti-KCa3.1 antibody (1:100, Alomone Labs, Jerusalem, Israel) or with a rabbit anti-AMPK-γ1 antibody (1:100 or 1:150, Abcam, Cambridge, MA).

    Techniques: Activity Assay, Patch Clamp, Expressing, Inhibition, Concentration Assay

    Membrane colocalization of HA-KCa3.1 channel and AMPK-γ1 in HEK-293 cells. A : immunostaining of HA-KCa3.1 channel performed on permeabilized HEK-293 cells expressing HA-KCa3.1 using a monoclonal anti-HA primary antibody plus an anti-rabbit antibody conjugated to AlexaFluor488 as secondary antibody. B : immunostaining of AMPK-γ1 performed on permeabilized HEK-293 cells expressing HA-KCa3.1 using an anti-AMPK-γ1 primary antibody plus an anti-rabbit antibody conjugated to AlexaFluor594 as secondary antibody. C : overlay of the AlexaFluor488 and AlexaFluor594 staining confirming the localization of AMPK-γ1 at the plasma membrane. Control experiments where no primary antibodies were added did not yield a detectable signal. Single optical sections were obtained by confocal fluorescence microscopy.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    doi: 10.1152/ajpcell.00418.2008

    Figure Lengend Snippet: Membrane colocalization of HA-KCa3.1 channel and AMPK-γ1 in HEK-293 cells. A : immunostaining of HA-KCa3.1 channel performed on permeabilized HEK-293 cells expressing HA-KCa3.1 using a monoclonal anti-HA primary antibody plus an anti-rabbit antibody conjugated to AlexaFluor488 as secondary antibody. B : immunostaining of AMPK-γ1 performed on permeabilized HEK-293 cells expressing HA-KCa3.1 using an anti-AMPK-γ1 primary antibody plus an anti-rabbit antibody conjugated to AlexaFluor594 as secondary antibody. C : overlay of the AlexaFluor488 and AlexaFluor594 staining confirming the localization of AMPK-γ1 at the plasma membrane. Control experiments where no primary antibodies were added did not yield a detectable signal. Single optical sections were obtained by confocal fluorescence microscopy.

    Article Snippet: For immunoprecipitation of endogenous KCa3.1 or AMPK-γ1 proteins, precleared soluble lysates were incubated for 1–2 h with a rabbit anti-KCa3.1 antibody (1:100, Alomone Labs, Jerusalem, Israel) or with a rabbit anti-AMPK-γ1 antibody (1:100 or 1:150, Abcam, Cambridge, MA).

    Techniques: Immunostaining, Expressing, Staining, Fluorescence, Microscopy

    Dominant negative approach supporting a functional link between AMPK-γ1 and KCa3.1. A1 : Western blot performed with an anti-Myc-tag antibody using HIK or HIK cells expressing the Myc-tagged dominant negative AMPK-γ1 R299G mutant. The presence of a band at 38 kDa confirms the expression of the AMPK-γ1 R299G mutant in HIK cells. A2 : Western blot where β-actin (Sigma, AC-15) was used as control for total protein loading with HIK cells and HIK cells expressing the AMPK-γ1 R299G mutant, respectively. B : inside-out patch-clamp recording of KCa3.1 performed using HIK cells. Recording obtained in 10 μM internal Ca 2+ conditions at an applied membrane potential of −60 mV. Perfusion with a 0 Ca 2+ solution (EGTA) is represented as a filled rectangle. Example of inside-out recording typical of 5/11 of HIK cells cotransfected with the dominant negative AMPK-γ1 mutant R299G. The mutation did not affect the channel activation by ATP but increased the fraction of the cells where KCa3.1 activity decreased by

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

    doi: 10.1152/ajpcell.00418.2008

    Figure Lengend Snippet: Dominant negative approach supporting a functional link between AMPK-γ1 and KCa3.1. A1 : Western blot performed with an anti-Myc-tag antibody using HIK or HIK cells expressing the Myc-tagged dominant negative AMPK-γ1 R299G mutant. The presence of a band at 38 kDa confirms the expression of the AMPK-γ1 R299G mutant in HIK cells. A2 : Western blot where β-actin (Sigma, AC-15) was used as control for total protein loading with HIK cells and HIK cells expressing the AMPK-γ1 R299G mutant, respectively. B : inside-out patch-clamp recording of KCa3.1 performed using HIK cells. Recording obtained in 10 μM internal Ca 2+ conditions at an applied membrane potential of −60 mV. Perfusion with a 0 Ca 2+ solution (EGTA) is represented as a filled rectangle. Example of inside-out recording typical of 5/11 of HIK cells cotransfected with the dominant negative AMPK-γ1 mutant R299G. The mutation did not affect the channel activation by ATP but increased the fraction of the cells where KCa3.1 activity decreased by

    Article Snippet: For immunoprecipitation of endogenous KCa3.1 or AMPK-γ1 proteins, precleared soluble lysates were incubated for 1–2 h with a rabbit anti-KCa3.1 antibody (1:100, Alomone Labs, Jerusalem, Israel) or with a rabbit anti-AMPK-γ1 antibody (1:100 or 1:150, Abcam, Cambridge, MA).

    Techniques: Dominant Negative Mutation, Functional Assay, Western Blot, Expressing, Mutagenesis, Patch Clamp, Activation Assay, Activity Assay

    Functional validation of KCNN4 mRNA copy number changes in detected human pyramidal cells. Waveforms of action potentials evoked by depolarizing current injections ( a ) and of extracellularly evoked EPSPs ( b ) responded differently to the serial application of the small- and intermediate-conductance calcium activated potassium channel activator NS309 (500 nM) and TRAM34 (1 μM), an inhibitor of intermediate-conductance calcium activated potassium channels. The descending phase of action potentials and EPSPs was shortened in pyramidal cells recorded in brain slices prepared from the Edema group, but remained unchanged in pyramidal neurons of the Control group and in fast spiking interneurons of the Edema group. Traces shown are population averages. Confocal images of immunoreactions with antibodies against Kcnn4 performed simultaneously on samples of the Control and Edema groups showing a cross section of the gray matter ( c ) and part of layer 3 similar to areas where electrophysiological experiments were performed ( d ). Pyramidal cells were not labeled in the Control group and moderate Kcnn4 positivity was detected in pyramidal cells (p) of the Edema group. In addition, intense immunolabeling for Kcnn4 was detected in glial cells resembling astrocytes and interlaminar glia in both groups of patients

    Journal: Acta Neuropathologica Communications

    Article Title: Human neuronal changes in brain edema and increased intracranial pressure

    doi: 10.1186/s40478-016-0356-x

    Figure Lengend Snippet: Functional validation of KCNN4 mRNA copy number changes in detected human pyramidal cells. Waveforms of action potentials evoked by depolarizing current injections ( a ) and of extracellularly evoked EPSPs ( b ) responded differently to the serial application of the small- and intermediate-conductance calcium activated potassium channel activator NS309 (500 nM) and TRAM34 (1 μM), an inhibitor of intermediate-conductance calcium activated potassium channels. The descending phase of action potentials and EPSPs was shortened in pyramidal cells recorded in brain slices prepared from the Edema group, but remained unchanged in pyramidal neurons of the Control group and in fast spiking interneurons of the Edema group. Traces shown are population averages. Confocal images of immunoreactions with antibodies against Kcnn4 performed simultaneously on samples of the Control and Edema groups showing a cross section of the gray matter ( c ) and part of layer 3 similar to areas where electrophysiological experiments were performed ( d ). Pyramidal cells were not labeled in the Control group and moderate Kcnn4 positivity was detected in pyramidal cells (p) of the Edema group. In addition, intense immunolabeling for Kcnn4 was detected in glial cells resembling astrocytes and interlaminar glia in both groups of patients

    Article Snippet: The tissue sections were incubated with Rabbit-KCNN4 1:300 (Alomone, APC-064) or Rabbit-KCNN4 1:100 (Thermo PA5-33875) primary antibody diluted in tris-buffered saline, 2 days at 4 °C.

    Techniques: Functional Assay, Labeling, Immunolabeling

    Functional expression of SK4 channels in breast cancer cells. (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Functional expression of SK4 channels in breast cancer cells. (A) Immunoblotting of SK4 and EMT-related proteins (E-cadherin and Vimentin) in breast cancer cell lines. (B) Comparison of SK4 mRNA expression in 4 breast cancer cell lines as determined by real-time PCR; n = 3. (C) Immunoblotting of ER protein in MDA-MB-468, MDA-MB-231 and T47D cells. (D-G) Immunostaining of SK4 (red) and nuclear marker DAPI (blue) in MDA-MB-231 (D), MDA-MB-468 (E), MCF-7 (F) and T47D (G) cells. Scale bars, 50 μm. (H, I) Whole-cell recording of MDA-MB-231 cells before (H) and after (I) 5-μM TRAM-34 treatment. (J, K) With (J) or without (K) 350 nM free Ca 2+ in the pipette solution, the voltage-current density curves show the currents changes before (a) and after (b) TRAM-34 treatment. The currents were evoked by step voltage ranging from -100 mV to +100 mV in steps of 10 mV every 100 ms. Dunnett’s Multiple Comparison Test was applied in comparison, ** p

    Article Snippet: Cells were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, blocked in 10% serum at 37°C for 30 min, and finally incubated with the SK4 antibody (1:20) (Alomone Labs, Israel) at 4°C overnight.

    Techniques: Functional Assay, Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Immunostaining, Marker, Transferring, Mass Spectrometry

    Blockage of SK4 channels inhibits MDA-MB-231 cell proliferation and colony formation ability, but not that of T47D cells. (A-D) Cell growth of MDA-MB-231 (A, B) and T47D cells (C, D) was analyzed using an MTT assay. The two cell lines were treated with 0–20 μM TRAM-34 (A, C) or clotrimazole (B, D) for 48 h, and the absorbance was measured; n = 5. (E, F) Images of the formed MDA-MB-231 colonies in the control group (CTL) and treatment groups (10 μM TRAM-34 and 20 μM TRAM-34); the bar represents separate counts of the colonies; n = 4. The data are presented as the mean ± SD, and Dunnett’s Multiple Comparison Test was applied in comparison. * p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Blockage of SK4 channels inhibits MDA-MB-231 cell proliferation and colony formation ability, but not that of T47D cells. (A-D) Cell growth of MDA-MB-231 (A, B) and T47D cells (C, D) was analyzed using an MTT assay. The two cell lines were treated with 0–20 μM TRAM-34 (A, C) or clotrimazole (B, D) for 48 h, and the absorbance was measured; n = 5. (E, F) Images of the formed MDA-MB-231 colonies in the control group (CTL) and treatment groups (10 μM TRAM-34 and 20 μM TRAM-34); the bar represents separate counts of the colonies; n = 4. The data are presented as the mean ± SD, and Dunnett’s Multiple Comparison Test was applied in comparison. * p

    Article Snippet: Cells were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, blocked in 10% serum at 37°C for 30 min, and finally incubated with the SK4 antibody (1:20) (Alomone Labs, Israel) at 4°C overnight.

    Techniques: Multiple Displacement Amplification, MTT Assay, CTL Assay

    Blockage of SK4 channels promotes apoptosis in MDA-MB-231 cells but not T47D cells. MDA-MB-231 (A, B) and T47D (C, D) cells were treated with 20 μM TRAM-34 for 24 or 48 h, and cell apoptosis was analyzed by Annexin V-FITC/ PI-PE staining and flow cytometry. The bar of MDA-MB-231 indicates that the apoptosis rate of the TRAM-34-treated group increased apparently compared with that of the control (CTL). For T47D, the difference was not significant. The data are presented as the mean ± SD, and unpaired t test was applied in comparison. n = 3; * p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Blockage of SK4 channels promotes apoptosis in MDA-MB-231 cells but not T47D cells. MDA-MB-231 (A, B) and T47D (C, D) cells were treated with 20 μM TRAM-34 for 24 or 48 h, and cell apoptosis was analyzed by Annexin V-FITC/ PI-PE staining and flow cytometry. The bar of MDA-MB-231 indicates that the apoptosis rate of the TRAM-34-treated group increased apparently compared with that of the control (CTL). For T47D, the difference was not significant. The data are presented as the mean ± SD, and unpaired t test was applied in comparison. n = 3; * p

    Article Snippet: Cells were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, blocked in 10% serum at 37°C for 30 min, and finally incubated with the SK4 antibody (1:20) (Alomone Labs, Israel) at 4°C overnight.

    Techniques: Multiple Displacement Amplification, Staining, Flow Cytometry, Cytometry, CTL Assay

    Down-regulation of SK4 channels inhibits the migration of MDA-MB-231 cells. A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: Down-regulation of SK4 channels inhibits the migration of MDA-MB-231 cells. A negative control siRNA (N.C.) and 3 SK4-specific siRNAs (Si-1, Si-2 and Si-3) were transfected into MDA-MB-231 cells, and 20 μM TRAM-34 was added to the TRAM-34-treated group to inhibit SK4 channels. (A, B) Knockdown of SK4 by siRNA was confirmed using immunoblotting and real-time PCR; n = 3. (C, D) The images and bar of the transwell migration assay indicate that the counts of migrated cells in SK4 siRNA (Si-SK4)- and TRAM-34-treated group were significantly less than those of the control (CTL). Scale bars, 50 μm; n = 4. (E, F) The images and bar of the wound-healing assay. The wound-healing rate represents the distance migrated by cells at certain time divided by the wound distance at 0 h. Scale bars, 100 μm; n = 3. The data are presented as the mean ± SD, Dunnett’s Multiple Comparison Test was applied in (B) and (D), and unpaired t test in (F). ** p

    Article Snippet: Cells were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, blocked in 10% serum at 37°C for 30 min, and finally incubated with the SK4 antibody (1:20) (Alomone Labs, Israel) at 4°C overnight.

    Techniques: Migration, Multiple Displacement Amplification, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Transwell Migration Assay, CTL Assay, Wound Healing Assay

    The EGF/bFGF-induced EMT of MDA-MB-231 cells correlates with SK4 channels. (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: The EGF/bFGF-induced EMT of MDA-MB-231 cells correlates with SK4 channels. (A) Phase contrast images of MDA-231 and T47D cells treated with (E+b) or without (CTL) EGF/bFGF for 1 day, 3 days and 5 days. Scale bars, 100 μm. (B, C) The EGF/bFGF-induced EMT of MDA-231 cells was confirmed using immunoblotting and real-time PCR of EMT markers (Vimentin, Snail1 and Slug), and the SK4 mRNA level increased after EMT. (D) Immunoblotting of EMT-related proteins (Vimentin and Snail1) was performed 72 h after MDA-231 cells were transfected with negative control siRNA (N.C.) or SK4-specific siRNA (Si-SK4); cells that did not undergo transfection served as a control (CTL). The data are presented as the mean ± SD, and paired t test was applied in comparison. n = 3; * p

    Article Snippet: Cells were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, blocked in 10% serum at 37°C for 30 min, and finally incubated with the SK4 antibody (1:20) (Alomone Labs, Israel) at 4°C overnight.

    Techniques: Multiple Displacement Amplification, CTL Assay, Real-time Polymerase Chain Reaction, Transfection, Negative Control

    SK4 proteins expressed in breast cancer tissue. (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).

    Journal: PLoS ONE

    Article Title: Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0154471

    Figure Lengend Snippet: SK4 proteins expressed in breast cancer tissue. (A-D) SK4 IHC in fours subtypes of breast cancer tissues including Luminal A (A), Luminal B (B), HER2 (C), and TNBC (D). Scale bars, 50 μm. (E) Immunoblotting of SK4 and E-cadherin in breast cancer tissues (BC1 and BC2) and non-tumor breast tissues (Non-Tumor1 and Non-Tumor2).

    Article Snippet: Cells were fixed with 1% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100, blocked in 10% serum at 37°C for 30 min, and finally incubated with the SK4 antibody (1:20) (Alomone Labs, Israel) at 4°C overnight.

    Techniques: Immunohistochemistry