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    Alomone Labs tram 34
    Potassium channels maintain plasma membrane polarization that is required for CPP entry into cells a , Assessment of the resting plasma membrane potential in the indicated wild-type cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence 10 μM XE-991 or <t>TRAM-34.</t> The grey and white zones correspond to non-treated cells and inhibitor-treated cells, respectively. NT, not treated. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. b , Effect of cellular depolarization (left of the grey zone) and hyperpolarization (right of the grey zone) on peptide internalization in the absence of serum. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 μg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 μM valinomycin), followed by the addition of TAT-RasGAP 317-326 for one hour. Alternatively, hyperpolarization was achieved by ectopic expression of the KCNJ2 potassium channel. Membrane potential and peptide internalization were then determined. Membrane potential was measured in the presence of DiBac4(3) by flow cytometry. Peptide internalization was measured by flow cytometry in the presence of 0.2% trypan blue. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. c , Quantitation of cytosolic CPP signal (top) and the number of endocytic vesicles per cell (bottom) in wild-type HeLa cells (n > 150 cells) incubated for one hour with 10 μM FITC-CPP in depolarizing (2 μg/ml gramicidin) or hyperpolarizing (10 μM valinomycin) conditions in the absence of serum based on confocal microscopy images (see Supplementary Fig. 9g ). Comparison between different conditions to non-treated control was done using ANOVA test with Dunnett’s correction for multiple comparison. The number of endocytic vesicles per cell was quantitated based on confocal images. Statistical comparison was done using t-tests. Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal. The confocal images were acquired in the middle of the cells based on Hoechst fluorescence. d , Internalization of various CPPs in the presence of different concentrations of potassium chloride in the media. Data for a given experiment are linked with thin blue lines.
    Tram 34, 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/tram 34/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    95
    Bruker Corporation 9 4t 105 mm bruker dmx spectrometer
    Potassium channels maintain plasma membrane polarization that is required for CPP entry into cells a , Assessment of the resting plasma membrane potential in the indicated wild-type cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence 10 μM XE-991 or <t>TRAM-34.</t> The grey and white zones correspond to non-treated cells and inhibitor-treated cells, respectively. NT, not treated. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. b , Effect of cellular depolarization (left of the grey zone) and hyperpolarization (right of the grey zone) on peptide internalization in the absence of serum. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 μg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 μM valinomycin), followed by the addition of TAT-RasGAP 317-326 for one hour. Alternatively, hyperpolarization was achieved by ectopic expression of the KCNJ2 potassium channel. Membrane potential and peptide internalization were then determined. Membrane potential was measured in the presence of DiBac4(3) by flow cytometry. Peptide internalization was measured by flow cytometry in the presence of 0.2% trypan blue. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. c , Quantitation of cytosolic CPP signal (top) and the number of endocytic vesicles per cell (bottom) in wild-type HeLa cells (n > 150 cells) incubated for one hour with 10 μM FITC-CPP in depolarizing (2 μg/ml gramicidin) or hyperpolarizing (10 μM valinomycin) conditions in the absence of serum based on confocal microscopy images (see Supplementary Fig. 9g ). Comparison between different conditions to non-treated control was done using ANOVA test with Dunnett’s correction for multiple comparison. The number of endocytic vesicles per cell was quantitated based on confocal images. Statistical comparison was done using t-tests. Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal. The confocal images were acquired in the middle of the cells based on Hoechst fluorescence. d , Internalization of various CPPs in the presence of different concentrations of potassium chloride in the media. Data for a given experiment are linked with thin blue lines.
    9 4t 105 Mm Bruker Dmx Spectrometer, supplied by Bruker Corporation, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/9 4t 105 mm bruker dmx spectrometer/product/Bruker Corporation
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    9 4t 105 mm bruker dmx spectrometer - by Bioz Stars, 2022-08
    95/100 stars
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    Image Search Results


    Potassium channels maintain plasma membrane polarization that is required for CPP entry into cells a , Assessment of the resting plasma membrane potential in the indicated wild-type cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence 10 μM XE-991 or TRAM-34. The grey and white zones correspond to non-treated cells and inhibitor-treated cells, respectively. NT, not treated. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. b , Effect of cellular depolarization (left of the grey zone) and hyperpolarization (right of the grey zone) on peptide internalization in the absence of serum. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 μg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 μM valinomycin), followed by the addition of TAT-RasGAP 317-326 for one hour. Alternatively, hyperpolarization was achieved by ectopic expression of the KCNJ2 potassium channel. Membrane potential and peptide internalization were then determined. Membrane potential was measured in the presence of DiBac4(3) by flow cytometry. Peptide internalization was measured by flow cytometry in the presence of 0.2% trypan blue. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. c , Quantitation of cytosolic CPP signal (top) and the number of endocytic vesicles per cell (bottom) in wild-type HeLa cells (n > 150 cells) incubated for one hour with 10 μM FITC-CPP in depolarizing (2 μg/ml gramicidin) or hyperpolarizing (10 μM valinomycin) conditions in the absence of serum based on confocal microscopy images (see Supplementary Fig. 9g ). Comparison between different conditions to non-treated control was done using ANOVA test with Dunnett’s correction for multiple comparison. The number of endocytic vesicles per cell was quantitated based on confocal images. Statistical comparison was done using t-tests. Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal. The confocal images were acquired in the middle of the cells based on Hoechst fluorescence. d , Internalization of various CPPs in the presence of different concentrations of potassium chloride in the media. Data for a given experiment are linked with thin blue lines.

    Journal: bioRxiv

    Article Title: Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Potassium channels maintain plasma membrane polarization that is required for CPP entry into cells a , Assessment of the resting plasma membrane potential in the indicated wild-type cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence 10 μM XE-991 or TRAM-34. The grey and white zones correspond to non-treated cells and inhibitor-treated cells, respectively. NT, not treated. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. b , Effect of cellular depolarization (left of the grey zone) and hyperpolarization (right of the grey zone) on peptide internalization in the absence of serum. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 μg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 μM valinomycin), followed by the addition of TAT-RasGAP 317-326 for one hour. Alternatively, hyperpolarization was achieved by ectopic expression of the KCNJ2 potassium channel. Membrane potential and peptide internalization were then determined. Membrane potential was measured in the presence of DiBac4(3) by flow cytometry. Peptide internalization was measured by flow cytometry in the presence of 0.2% trypan blue. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis with Dunnett’s correction. Each dot in a given condition represents an independent experiment. c , Quantitation of cytosolic CPP signal (top) and the number of endocytic vesicles per cell (bottom) in wild-type HeLa cells (n > 150 cells) incubated for one hour with 10 μM FITC-CPP in depolarizing (2 μg/ml gramicidin) or hyperpolarizing (10 μM valinomycin) conditions in the absence of serum based on confocal microscopy images (see Supplementary Fig. 9g ). Comparison between different conditions to non-treated control was done using ANOVA test with Dunnett’s correction for multiple comparison. The number of endocytic vesicles per cell was quantitated based on confocal images. Statistical comparison was done using t-tests. Quantitation of vesicles was not performed in hyperpolarizing conditions due to masking from strong cytosolic signal. The confocal images were acquired in the middle of the cells based on Hoechst fluorescence. d , Internalization of various CPPs in the presence of different concentrations of potassium chloride in the media. Data for a given experiment are linked with thin blue lines.

    Article Snippet: XE-991 and TRAM-34 (Alomone labs, ref. no. X-100 and T-105 respectively) was dissolved in DMSO at 100 mM and stored at -20 °C.

    Techniques: Knock-Out, Expressing, Flow Cytometry, Quantitation Assay, Incubation, Confocal Microscopy, Fluorescence

    Identification of potassium channels as mediators of direct translocation of CPPs into cells a , Identification of genes implicated in TAT-RasGAP 317-326 internalization in Raji and SKW6.4 cells. The graphs depict the p-value (calculated using the MAGeCK procedure; see Materials and Methods) for the difference in sgRNA expression between peptide-treated and control cells for the ∼20’000 genes targeted by the CRISPR/Cas9 library. b , Quantitation of TAT-RasGAP 317-326 entry (top) and induced death (bottom) in wild-type (WT) and knock-out (KO) cells. The WT and the corresponding potassium channel KO versions of the indicated cell lines were pretreated or not for 30 minutes with 10 μM XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 μM (Raji and SKW6.4 cells) or 80 μM (HeLa cells) TAT-RasGAP 317-326 . Internalization was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. TAT-RasGAP 317-326 concentrations and time of incubation used were adjusted so that the CPP induced similar cell death (between 60% and 90%) in the wild-type versions of the different cell lines. c , Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The high percentage of cells with vesicular staining in the knock-out (KO) cells results from the absence of strong diffuse staining masking endosomes. The results correspond to the average of three experiments.

    Journal: bioRxiv

    Article Title: Genetic, cellular and structural characterization of the membrane potential-dependent cell-penetrating peptide translocation pore

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells a , Identification of genes implicated in TAT-RasGAP 317-326 internalization in Raji and SKW6.4 cells. The graphs depict the p-value (calculated using the MAGeCK procedure; see Materials and Methods) for the difference in sgRNA expression between peptide-treated and control cells for the ∼20’000 genes targeted by the CRISPR/Cas9 library. b , Quantitation of TAT-RasGAP 317-326 entry (top) and induced death (bottom) in wild-type (WT) and knock-out (KO) cells. The WT and the corresponding potassium channel KO versions of the indicated cell lines were pretreated or not for 30 minutes with 10 μM XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 μM (Raji and SKW6.4 cells) or 80 μM (HeLa cells) TAT-RasGAP 317-326 . Internalization was recorded after one hour and cell death after 16 hours (Raji and SKW6.4) or 24 hours (HeLa). Results correspond to the average of three independent experiments. TAT-RasGAP 317-326 concentrations and time of incubation used were adjusted so that the CPP induced similar cell death (between 60% and 90%) in the wild-type versions of the different cell lines. c , Quantitation of the modalities of TAT-RasGAP 317-326 entry in wild-type and KCNQ5 knock-out Raji cells. Cells were incubated with FITC-TAT-RasGAP 317-326 for various periods of time and peptide staining was visually quantitated on confocal images (n > 150 cells for each time-point). The high percentage of cells with vesicular staining in the knock-out (KO) cells results from the absence of strong diffuse staining masking endosomes. The results correspond to the average of three experiments.

    Article Snippet: XE-991 and TRAM-34 (Alomone labs, ref. no. X-100 and T-105 respectively) was dissolved in DMSO at 100 mM and stored at -20 °C.

    Techniques: Translocation Assay, Expressing, CRISPR, Quantitation Assay, Knock-Out, Incubation, Staining

    Ovariectomy reduces SK3 channel current density in endothelial cells. A : Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B : SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C : Representative whole-cell current density obtained from ovx endothelial cells. D : SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E : Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F : Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P

    Journal: PLoS ONE

    Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

    doi: 10.1371/journal.pone.0104686

    Figure Lengend Snippet: Ovariectomy reduces SK3 channel current density in endothelial cells. A : Representative traces recorded using conventional whole-cell recording on endothelial cells isolated from mesenteric arteries obtained from control mouse. Cells were voltage clamped at their resting membrane potential and a 200 ms voltage ramp from of −80 to +60 mV was delivered to elicit whole cell currents before (control) and after subsequent bath application of apamin (apamin) and apamin+tram34 (tram34). B : SK3 and IK1 current densities isolated from digital subtraction of the traces shown in (A) for control endothelial cells. C : Representative whole-cell current density obtained from ovx endothelial cells. D : SK3 and IK1 current densities isolated from digital subtraction of the traces in (C) for ovx endothelial cells. E : Summarized whole-cell SK3 and IK1 current densities from control (black) and ovx (grey) endothelial cells measured at +30 mV. F : Normalized SK3/IK1 ratios for control (black) and ovx (grey) recordings showing reduced SK3 channel activity in ovx endothelial cells. Asterisk (*) indicates statistical significance from control (P

    Article Snippet: Tram34 was from Alomone Labs (Israel).

    Techniques: Isolation, Mass Spectrometry, Activity Assay

    Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels. A : (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B : Representative force myograph trace obtained from an ovx artery. C and D : Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their ( C ) change in tone and ( D ) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C : Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D : Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI 2 ) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.

    Journal: PLoS ONE

    Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

    doi: 10.1371/journal.pone.0104686

    Figure Lengend Snippet: Reduced ACh-induced vasorelaxation due to decreased SK3 channel contribution in ovx vessels. A : (left panel) Representative force myograph recording showing tension (mN) plotted against time (s) using a mesenteric vessel obtained from control mouse. Addition of 3 µM PE increased tension and 1 µM ACh caused 64% vasorelaxation, normalized to the PE-induced tension. (right panel) Following bath washout, PE was added to pre-contract the vessel ∼50%, followed by the addition of 100 µM L-NAME and 1 µM ACh. L-NAME-induced 61% increase in PE-induced contraction and ACh reduced tension by 34%. B : Representative force myograph trace obtained from an ovx artery. C and D : Summarized results for (A and B) and for other selective inhibitors to block different vasorelaxation pathways to study their ( C ) change in tone and ( D ) contribution to ACh-induced relaxation for both control (black bars) and ovx (grey bars) vessels. C : Change in tone was obtained from tension increase in the presence of inhibitors normalized to the baseline tension (eg. 61% and 34% increase in the presence of L-NAME for control and ovx vessels, respectively, as shown in A and B). D : Contribution to ACh-induced relaxation was calculated from the difference in ACh relaxation before and after inhibitor treatment, normalized to the control (before) ACh relaxation. L-NAME blocks nitric oxide (NO) pathway; indomethacin blocks prostacyclin (PGI 2 ) pathway; apamin (apa) and tram34 (tram) together block the EDH pathway.

    Article Snippet: Tram34 was from Alomone Labs (Israel).

    Techniques: Blocking Assay

    IK1 channel activity mediates TRPV4-induced vasorelaxation in ovx vessels. A : Summarized results from studies using 500 nM HC067047 (HC), a TRPV4 channel antagonist, on change in tone (left panel) and contribution to ACh-induced relaxation (right panel) using vessels obtained from both control (black) and ovx (grey) mice. B : Summarized results from studies using 300 nM GSK1016790, a TRPV4 channel agonist, on changes to vascular tension in the absence and presence of apamin (apa) and/or tram34 (tram). These studies were performed in the presence of L-NAME and indomethacin. Asterisk (*) denotes statistical significance (P

    Journal: PLoS ONE

    Article Title: Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

    doi: 10.1371/journal.pone.0104686

    Figure Lengend Snippet: IK1 channel activity mediates TRPV4-induced vasorelaxation in ovx vessels. A : Summarized results from studies using 500 nM HC067047 (HC), a TRPV4 channel antagonist, on change in tone (left panel) and contribution to ACh-induced relaxation (right panel) using vessels obtained from both control (black) and ovx (grey) mice. B : Summarized results from studies using 300 nM GSK1016790, a TRPV4 channel agonist, on changes to vascular tension in the absence and presence of apamin (apa) and/or tram34 (tram). These studies were performed in the presence of L-NAME and indomethacin. Asterisk (*) denotes statistical significance (P

    Article Snippet: Tram34 was from Alomone Labs (Israel).

    Techniques: Activity Assay, Mouse Assay