k252a  (Alomone Labs)


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    Structured Review

    Alomone Labs k252a
    A – D , Effect of <t>K252a</t> and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.
    K252a, 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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    k252a - by Bioz Stars, 2022-07
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    Images

    1) Product Images from "Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells"

    Article Title: Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-22-08915.2001

    A – D , Effect of K252a and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.
    Figure Legend Snippet: A – D , Effect of K252a and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.

    Techniques Used: Activity Assay, SDS Page, Purification, Labeling, Molecular Weight

    Internalization of 125 I-NT-3 (10–20 ng/ml) in purified retinal ganglion cells from E18–21 chick embryos does not require tyrosine kinase activity. The amount of internalization in the presence of 1 μg/ml K252a (tyrosine kinase inhibitor) is plotted as the percentage relative to the values for vehicle (1 μg/ml DMSO), which were averaged to 100% internalization (∼40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C) is indicated ( dotted line ). Error bars indicate SEM. The number of independent experiments (each in duplicate) is indicated. The p level for confidence ( t test) is indicated, showing no significant effect of K252a on NT-3 internalization.
    Figure Legend Snippet: Internalization of 125 I-NT-3 (10–20 ng/ml) in purified retinal ganglion cells from E18–21 chick embryos does not require tyrosine kinase activity. The amount of internalization in the presence of 1 μg/ml K252a (tyrosine kinase inhibitor) is plotted as the percentage relative to the values for vehicle (1 μg/ml DMSO), which were averaged to 100% internalization (∼40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C) is indicated ( dotted line ). Error bars indicate SEM. The number of independent experiments (each in duplicate) is indicated. The p level for confidence ( t test) is indicated, showing no significant effect of K252a on NT-3 internalization.

    Techniques Used: Purification, Activity Assay

    Diagram summarizing proposed subcellular pathways of internalized NT-3 ( black dots ) in a retinal ganglion cell. At least two pathways of internalized NT-3 can be distinguished. A lysosomal pathway of NT-3 may be common to all neurotrophins and may involve binding to the p75 receptor ( U ) or binding of NT-3 to trkC receptor ( Y ) in the presence of the tyrosine kinase inhibitor K252a. The neurotrophin is degraded in lysosomes ( LYS ). Alternatively, a novel pathway of NT-3 internalized in endosomes fuses with membranes of the Golgi apparatus: Golgi Pathway . This sorting requires tyrosine kinase activity (presumably trkC , Y ), and this pathway may join that of newly synthesized neurotrophins as well as p75 receptor ( U ) from the endoplasmic reticulum ( ER ). After passage through the Golgi system, internalized NT-3 is packaged in presumptive large dense-core vesicles ( LDCV ) for anterograde axonal transport. In this pathway, internalized NT-3 binds preferentially to p75. Anterogradely transported NT-3 is released from RGC axon terminals in the tectum, and after release it binds predominantly to trkC in tectal cells where it accumulates in multivesicular bodies ( MVB ).
    Figure Legend Snippet: Diagram summarizing proposed subcellular pathways of internalized NT-3 ( black dots ) in a retinal ganglion cell. At least two pathways of internalized NT-3 can be distinguished. A lysosomal pathway of NT-3 may be common to all neurotrophins and may involve binding to the p75 receptor ( U ) or binding of NT-3 to trkC receptor ( Y ) in the presence of the tyrosine kinase inhibitor K252a. The neurotrophin is degraded in lysosomes ( LYS ). Alternatively, a novel pathway of NT-3 internalized in endosomes fuses with membranes of the Golgi apparatus: Golgi Pathway . This sorting requires tyrosine kinase activity (presumably trkC , Y ), and this pathway may join that of newly synthesized neurotrophins as well as p75 receptor ( U ) from the endoplasmic reticulum ( ER ). After passage through the Golgi system, internalized NT-3 is packaged in presumptive large dense-core vesicles ( LDCV ) for anterograde axonal transport. In this pathway, internalized NT-3 binds preferentially to p75. Anterogradely transported NT-3 is released from RGC axon terminals in the tectum, and after release it binds predominantly to trkC in tectal cells where it accumulates in multivesicular bodies ( MVB ).

    Techniques Used: Binding Assay, Activity Assay, Synthesized

    A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Activity Assay, Injection, Blocking Assay, Derivative Assay

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    Alomone Labs sk4 antibody
    Functional expression of <t>SK4</t> 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
    Sk4 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Alomone Labs anti bk α subunit primary antibody
    BK channel protein levels are not altered by diet. A : the anti-BK <t>α-subunit</t> antibody detected a single protein band at ∼120 kDa consistent with previously published molecular weights. This band was eliminated by preincubating the primary
    Anti Bk α Subunit Primary Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti bk α subunit primary antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Alomone Labs bk α subunit ab
    Immunohistochemical identification of BK in a cannulated mesenteric venule taken from a control animal. Small mesenteric venules were cannulated and pressurized (∼10 mmHg) as described in the text. BK was identified with an antibody (Alomone APC-107) directed against the <t>α-subunit</t> (green). Nuclei were stained with propidium iodide (red) to assist in identification of cells. Images of cannulated vessels were taken using confocal microscopy. A : marked BK staining in vascular smooth muscle cells. B : comparatively less intense, but positive, staining for BK in endothelial cells (arrows). Nuclear staining confirms cellular orientation consistent with endothelial cells. Image represents a composite 10 Z -stack images taken at 0.1-μm distances. C ) using Western blotting have confirmed specificity of the primary antibody. D : enlargment of the area indicated by the dashed rectangle overlying B . Endothelial staining for BK was obtained in 6 additional preparations processed in a similar manner.
    Bk α Subunit Ab, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bk α subunit ab/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bk α subunit ab - by Bioz Stars, 2022-07
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    Image Search Results


    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

    BK channel protein levels are not altered by diet. A : the anti-BK α-subunit antibody detected a single protein band at ∼120 kDa consistent with previously published molecular weights. This band was eliminated by preincubating the primary

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: High-fat diet-induced obesity alters nitric oxide-mediated neuromuscular transmission and smooth muscle excitability in the mouse distal colon

    doi: 10.1152/ajpgi.00085.2016

    Figure Lengend Snippet: BK channel protein levels are not altered by diet. A : the anti-BK α-subunit antibody detected a single protein band at ∼120 kDa consistent with previously published molecular weights. This band was eliminated by preincubating the primary

    Article Snippet: Membranes were incubated for 2 h with anti-BK α-subunit primary antibody (1:20,000 dilution; APC-107; Alomone Labs, Jerusalem, Israel).

    Techniques:

    Immunohistochemical identification of BK in a cannulated mesenteric venule taken from a control animal. Small mesenteric venules were cannulated and pressurized (∼10 mmHg) as described in the text. BK was identified with an antibody (Alomone APC-107) directed against the α-subunit (green). Nuclei were stained with propidium iodide (red) to assist in identification of cells. Images of cannulated vessels were taken using confocal microscopy. A : marked BK staining in vascular smooth muscle cells. B : comparatively less intense, but positive, staining for BK in endothelial cells (arrows). Nuclear staining confirms cellular orientation consistent with endothelial cells. Image represents a composite 10 Z -stack images taken at 0.1-μm distances. C ) using Western blotting have confirmed specificity of the primary antibody. D : enlargment of the area indicated by the dashed rectangle overlying B . Endothelial staining for BK was obtained in 6 additional preparations processed in a similar manner.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of BK channels

    doi: 10.1152/ajpheart.01229.2009

    Figure Lengend Snippet: Immunohistochemical identification of BK in a cannulated mesenteric venule taken from a control animal. Small mesenteric venules were cannulated and pressurized (∼10 mmHg) as described in the text. BK was identified with an antibody (Alomone APC-107) directed against the α-subunit (green). Nuclei were stained with propidium iodide (red) to assist in identification of cells. Images of cannulated vessels were taken using confocal microscopy. A : marked BK staining in vascular smooth muscle cells. B : comparatively less intense, but positive, staining for BK in endothelial cells (arrows). Nuclear staining confirms cellular orientation consistent with endothelial cells. Image represents a composite 10 Z -stack images taken at 0.1-μm distances. C ) using Western blotting have confirmed specificity of the primary antibody. D : enlargment of the area indicated by the dashed rectangle overlying B . Endothelial staining for BK was obtained in 6 additional preparations processed in a similar manner.

    Article Snippet: The vessels were then fixed with 2% paraformaldehyde for 20 min and stained with primary BK-α subunit Ab [rabbit anti-mouse Ab (1:50); Alomone Labs, Israel], followed by goat-anti rabbit IgG (1:100; Alexa fluor conjugated; 488 nm excitation wavelength).

    Techniques: Immunohistochemistry, Staining, Confocal Microscopy, Western Blot