tram 34  (Alomone Labs)


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    Name:
    TRAM 34
    Description:
    TRAM 34 is a potent and selective KCa3 1 SK4 intermediate conductance Ca2 activated K channel inhibitor As such it is a powerful pharmacological tool that enables the determination of the roles played by KCa3 1 channels in different physiological systems ranging from activation of T lymphocytes to migration of endothelial cells
    Catalog Number:
    T-105
    Price:
    67.0
    Category:
    Small Molecule
    Source:
    Synthetic
    Applications:
    0
    Purity:
    >99%
    Size:
    5 mg
    Format:
    Lyophilized/solid.
    Formula:
    C22H17ClN2
    Molecular Weight:
    344.8
    Molecule Name:
    1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole.
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    Structured Review

    Alomone Labs tram 34
    TRAM 34
    TRAM 34 is a potent and selective KCa3 1 SK4 intermediate conductance Ca2 activated K channel inhibitor As such it is a powerful pharmacological tool that enables the determination of the roles played by KCa3 1 channels in different physiological systems ranging from activation of T lymphocytes to migration of endothelial cells
    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
    tram 34 - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells"

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.963017

    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.

    Techniques Used: Translocation Assay, Knock-Out, Quantitation Assay, Flow Cytometry, Incubation, Fluorescence, Binding Assay

    Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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 XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake 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. (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 increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.
    Figure Legend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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 XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake 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. (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 increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.

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

    Related to Fig. 2. Potassium channels regulate the cellular uptake of various TAT-bound cargos. (A) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent experiments.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels regulate the cellular uptake of various TAT-bound cargos. (A) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent experiments.

    Techniques Used: Luciferase, Activity Assay, Microscopy, Knock-Out, Expressing

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.
    Figure Legend 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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.

    Techniques Used: Knock-Out, Expressing, Quantitation Assay, Flow Cytometry, Two Tailed Test

    2) Product Images from "Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells"

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.25.963017

    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.

    Techniques Used: Translocation Assay, Knock-Out, Quantitation Assay, Flow Cytometry, Incubation, Fluorescence, Binding Assay

    Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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 XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake 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. (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 increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.
    Figure Legend Snippet: Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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 XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake 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. (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 increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.

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

    Related to Fig. 2. Potassium channels regulate the cellular uptake of various TAT-bound cargos. (A) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent experiments.
    Figure Legend Snippet: Related to Fig. 2. Potassium channels regulate the cellular uptake of various TAT-bound cargos. (A) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent experiments.

    Techniques Used: Luciferase, Activity Assay, Microscopy, Knock-Out, Expressing

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.
    Figure Legend 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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.

    Techniques Used: Knock-Out, Expressing, Quantitation Assay, Flow Cytometry, Two Tailed Test

    Related Articles

    other:

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells
    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.

    Article Title: Overexpression of Large-Conductance Calcium-Activated Potassium Channels in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration.
    Article Snippet: TRAM-34, NS169, and paxilline were prepared in DMSO (at concentrations of 10, 10, and 5mM, respectively).

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells
    Article Snippet: Blasticidin (Applichem, ref. no. A3784) was dissolved at 1 mg/mL in water and stored at −20 °C.

    Blocking Assay:

    Article Title: Functional role of the KCa3.1 potassium channel in synovial fibroblasts from rheumatoid arthritis patients.
    Article Snippet: .. Rheumatoid arthritis synovial fibroblasts (RA-SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. ..

    Patch Clamp:

    Article Title: Functional role of the KCa3.1 potassium channel in synovial fibroblasts from rheumatoid arthritis patients.
    Article Snippet: .. Rheumatoid arthritis synovial fibroblasts (RA-SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. ..

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    Alomone Labs tram 34
    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or <t>TRAM-34</t> (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.
    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
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    tram 34 - by Bioz Stars, 2021-09
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    Alomone Labs thapsigargin
    MTII-LRP-mediated chemoattraction requires the activation of calcium signaling and co-receptors within the growth cone. A , reducing the concentration of extracellular calcium (low [ Ca 2 + ] EC ) reversed growth cone turning in response to MTII so that growth cones were repulsed by a microgradient of MTII. Depletion of intracellular calcium stores with <t>thapsigargin</t> abolished turning in response to MTII. The inhibitor of CaMKII, KN93, reversed turning in response to MTII so that growth cones were repulsed by a microgradient of MTII, whereas the inactive analogue KN92 had no effect on turning. B , inhibition of TrkA was shown to abolish growth cone turning in response to MTII. Inhibition of TrkA and other kinases by K252a reversed turning from attraction to repulsion. Specific inhibition of TrkA with GW441756 or a TrkA antibody abolished turning in response to MTII so that the turning angle did not differ from random control growth. C , representative immunocytochemistry images of individual growth cones turning in response to microgradients of vehicle (PBS) or MTII. Growth cones were rapidly fixed during turning and stained for TrKA ( red ) or phosphorylated TrKA ( pTrKA , blue ) and actin ( green ). The actin labeling was used to depict the growth cone area, and the growth cones were divided into near and far regions with respect to the micropipette for pixel intensity analysis. The dotted line drawn from the axon through the growth cone separates the near and far regions of the growth cone. D , quantification of total TrkA and phosphorylated TrkA expression localized to the near versus far side of the growth cone while turning toward a gradient of MTII. *** and ###, p
    Thapsigargin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs ttx
    MTII-LRP-mediated chemoattraction requires the activation of calcium signaling and co-receptors within the growth cone. A , reducing the concentration of extracellular calcium (low [ Ca 2 + ] EC ) reversed growth cone turning in response to MTII so that growth cones were repulsed by a microgradient of MTII. Depletion of intracellular calcium stores with <t>thapsigargin</t> abolished turning in response to MTII. The inhibitor of CaMKII, KN93, reversed turning in response to MTII so that growth cones were repulsed by a microgradient of MTII, whereas the inactive analogue KN92 had no effect on turning. B , inhibition of TrkA was shown to abolish growth cone turning in response to MTII. Inhibition of TrkA and other kinases by K252a reversed turning from attraction to repulsion. Specific inhibition of TrkA with GW441756 or a TrkA antibody abolished turning in response to MTII so that the turning angle did not differ from random control growth. C , representative immunocytochemistry images of individual growth cones turning in response to microgradients of vehicle (PBS) or MTII. Growth cones were rapidly fixed during turning and stained for TrKA ( red ) or phosphorylated TrKA ( pTrKA , blue ) and actin ( green ). The actin labeling was used to depict the growth cone area, and the growth cones were divided into near and far regions with respect to the micropipette for pixel intensity analysis. The dotted line drawn from the axon through the growth cone separates the near and far regions of the growth cone. D , quantification of total TrkA and phosphorylated TrkA expression localized to the near versus far side of the growth cone while turning toward a gradient of MTII. *** and ###, p
    Ttx, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent experiments.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Related to Fig. 2. Potassium channels modulate direct CPP translocation, but not endocytosis. (A) Same as Fig. 2C , but for wild-type and KCNN4 and KCNK5 SKW6.4 knock-out cells. The results correspond to the average of three independent experiments. (B) As in panel A, but for wild-type and KCNN4 knock-out HeLa cells. The results correspond to the average of three independent experiments. (C) Quantitation of TAT-RasGAP 317-326 cytosolic access resulting from direct translocation (left, n=19) and endosomal escape (middle, n=16 and right. n=22) in the presence or in the absence of 1 mM LLOME, a endosome/lysosome disruptor( 36 ). In direct translocation condition, the peptide was continuously present in the media, while it was washed out after 30 minutes to assess endosomal escape. LLOME was used to show that our experimental setup allows for the detection of cytoplasmic CPPs if they are released from cellular vesicles. The results correspond to three independent experiments. (D) Quantitation by flow cytometry of 20 mg/ml AlexaFluor488-transferrin uptake in the wild-type (WT) cell lines and the corresponding knock-out (KO) cells, pretreated or not for 30 minutes with the XE-991 (10 mM) or TRAM-34 (10 mM) potassium channel inhibitors. Transferrin uptake was allowed to proceed for 60 minutes (still in the presence of inhibitors when these were used in the 30 minute pre-incubation period). To quench membrane bound transferrin fluorescence, cells were incubated with 0.2% trypan blue prior to flow cytometry analysis (see Fig. S1F-G ). The independent experiment replicates are color-coded. (E) Assessment of FITC-TAT-RasGAP 317-326 binding to cellular membrane of wild-type and KCNQ5 knock-out Raji cells after 60 seconds of incubation (top), as well as associated peptide uptake after one hour of treatment (bottom). The results correspond to at least five independent 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, Knock-Out, Quantitation Assay, Flow Cytometry, Incubation, Fluorescence, Binding Assay

    Identification of potassium channels as mediators of direct translocation of CPPs into cells. (A) Identification of genes implicated in TAT-RasGAP 317-326 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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 XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake 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. (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 increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. The results correspond to the average of three experiments.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    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 uptake in Raji cells (upper panel) and SKW6.4 cells (lower panel). 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 XE-991 or with TRAM-34 and then incubated (still in the presence of the inhibitors when initially added) with, or without 40 mM (Raji and SKW6.4 cells) or 80 mM (HeLa cells) TAT-RasGAP 317-326 . Uptake 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. (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 increased percentage of cells with vesicular staining in the KO cells is likely the result of unmasking from the disappearance of strong diffuse staining. 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

    Related to Fig. 2. Potassium channels regulate the cellular uptake of various TAT-bound cargos. (A) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent experiments.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    doi: 10.1101/2020.02.25.963017

    Figure Lengend Snippet: Related to Fig. 2. Potassium channels regulate the cellular uptake of various TAT-bound cargos. (A) TAT-PNA-induced luciferase activity in the indicated cell lines pretreated or not with potassium channel inhibitors (XE-991 or TRAM-34) or genetically invalidated for specific potassium channels. Results are normalized to non-stimulated cells (dashed lines). The independent experiment replicates are color-coded. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. (B) Representative microscopy images of wild-type and KCNQ5 knock-out Raji cells expressing loxP-RFP-STOP-loxP-GFP and treated or not with 20 mM TAT-Cre for 48 hours. The results correspond to one of three independent experiments. (C) Uptake of FITC-D-JNKI1 in the indicated cell lines genetically invalidated (KO) or not (WT) for specific potassium channels. The results correspond to the median of three independent 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: Luciferase, Activity Assay, Microscopy, Knock-Out, Expressing

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. Data for a given experiment are linked with thin blue lines.

    Journal: bioRxiv

    Article Title: Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

    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 cell lines and the corresponding potassium channel knock-out (KO) clones in the presence or in the absence of 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. 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 uptake. The indicated cell lines and the corresponding channel knock-out (KO) clones were pretreated or not with depolarization agents (2 mg/ml gramicidin for 5 minutes or high extracellular potassium buffer for 30 minutes) or with hyperpolarization inducer (10 mM 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 uptake were then determined. The p-values correspond to the assessment of the significance of the differences with the control wild-type condition using ANOVA multiple comparison analysis. Each dot in a given condition represents an independent experiment. (C) Quantitation of CPP uptake in Raji cells by flow cytometry in depolarizing (top) or hyperpolarizing (bottom) conditions. Data from a given independent experiment are connected by lines. Comparison between two conditions was done using two-tailed paired t-test. (D) Uptake of various CPPs in the presence of different concentrations of potassium chloride in the media. The thick grey lines correspond to the average of 3-5 independent experiments. 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, Quantitation Assay, Flow Cytometry, Two Tailed Test

    MTII-LRP-mediated chemoattraction requires the activation of calcium signaling and co-receptors within the growth cone. A , reducing the concentration of extracellular calcium (low [ Ca 2 + ] EC ) reversed growth cone turning in response to MTII so that growth cones were repulsed by a microgradient of MTII. Depletion of intracellular calcium stores with thapsigargin abolished turning in response to MTII. The inhibitor of CaMKII, KN93, reversed turning in response to MTII so that growth cones were repulsed by a microgradient of MTII, whereas the inactive analogue KN92 had no effect on turning. B , inhibition of TrkA was shown to abolish growth cone turning in response to MTII. Inhibition of TrkA and other kinases by K252a reversed turning from attraction to repulsion. Specific inhibition of TrkA with GW441756 or a TrkA antibody abolished turning in response to MTII so that the turning angle did not differ from random control growth. C , representative immunocytochemistry images of individual growth cones turning in response to microgradients of vehicle (PBS) or MTII. Growth cones were rapidly fixed during turning and stained for TrKA ( red ) or phosphorylated TrKA ( pTrKA , blue ) and actin ( green ). The actin labeling was used to depict the growth cone area, and the growth cones were divided into near and far regions with respect to the micropipette for pixel intensity analysis. The dotted line drawn from the axon through the growth cone separates the near and far regions of the growth cone. D , quantification of total TrkA and phosphorylated TrkA expression localized to the near versus far side of the growth cone while turning toward a gradient of MTII. *** and ###, p

    Journal: The Journal of Biological Chemistry

    Article Title: Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration *

    doi: 10.1074/jbc.M115.668996

    Figure Lengend Snippet: MTII-LRP-mediated chemoattraction requires the activation of calcium signaling and co-receptors within the growth cone. A , reducing the concentration of extracellular calcium (low [ Ca 2 + ] EC ) reversed growth cone turning in response to MTII so that growth cones were repulsed by a microgradient of MTII. Depletion of intracellular calcium stores with thapsigargin abolished turning in response to MTII. The inhibitor of CaMKII, KN93, reversed turning in response to MTII so that growth cones were repulsed by a microgradient of MTII, whereas the inactive analogue KN92 had no effect on turning. B , inhibition of TrkA was shown to abolish growth cone turning in response to MTII. Inhibition of TrkA and other kinases by K252a reversed turning from attraction to repulsion. Specific inhibition of TrkA with GW441756 or a TrkA antibody abolished turning in response to MTII so that the turning angle did not differ from random control growth. C , representative immunocytochemistry images of individual growth cones turning in response to microgradients of vehicle (PBS) or MTII. Growth cones were rapidly fixed during turning and stained for TrKA ( red ) or phosphorylated TrKA ( pTrKA , blue ) and actin ( green ). The actin labeling was used to depict the growth cone area, and the growth cones were divided into near and far regions with respect to the micropipette for pixel intensity analysis. The dotted line drawn from the axon through the growth cone separates the near and far regions of the growth cone. D , quantification of total TrkA and phosphorylated TrkA expression localized to the near versus far side of the growth cone while turning toward a gradient of MTII. *** and ###, p

    Article Snippet: The bath-applied pharmacological agents were as follows: RAP (25 μg/ml), Tris-buffered saline, KN93 (5 μm , Calbiochem), KN92 (5 μm , Calbiochem), thapsigargin (50 nm , Alomone Labs, Jerusalem, Israel), K252a (100 nm, Calbiochem), and GW441756 (Tocris Bioscience).

    Techniques: Activation Assay, Concentration Assay, Inhibition, Immunocytochemistry, Staining, Labeling, Expressing