anti pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 4 5 p 2
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN"

    Article Title: Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-024-06779-3

    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Figure Legend Snippet: A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.

    Techniques Used: Western Blot, Plasmid Preparation, Over Expression, Activation Assay

    anti pi 4 5 p 2 p z045  (Echelon Biosciences)


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    Echelon Biosciences anti pi 4 5 p 2 p z045
    Anti Pi 4 5 P 2 P Z045, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 4 5 p 2
    Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 4 5 p 2
    Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pi 4 5 p 2 biotinylated antibody  (Echelon Biosciences)


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    Echelon Biosciences anti pi 4 5 p 2 biotinylated antibody
    Anti Pi 4 5 P 2 Biotinylated Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pi 4 5 p 2 biotinylated antibody  (Echelon Biosciences)


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    Echelon Biosciences anti pi 4 5 p 2 biotinylated antibody
    Anti Pi 4 5 P 2 Biotinylated Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pi 4 5 p 2 biotinylated antibody  (Echelon Biosciences)


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    Echelon Biosciences anti pi 4 5 p 2 biotinylated antibody
    Anti Pi 4 5 P 2 Biotinylated Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pi 4 5 p 2 antibody  (Echelon Biosciences)


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    Echelon Biosciences pi 4 5 p 2 antibody
    Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.
    Pi 4 5 P 2 Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane"

    Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2023.104812

    Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.
    Figure Legend Snippet: Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.

    Techniques Used: Translocation Assay, Confocal Microscopy, Transfection, Over Expression, Fluorescence, Binding Assay

    OCRL-ORP4L interaction is required for PI(4,5)P 2 consumption in the PM. A , Western blot showing OCRL expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . Cells were subjected to control (shNT), OCRL1 knockdown (sh OCRL1 ) alone, or combined with OCRL1 rescued expression (sh OCRL1 + OCRL1 ) or combined with OCRL1 (Mut) rescued expression (sh OCRL1 + OCRL1 (Mut)). Cells were transduced with lentivirus expressing shNT or sh OCRL1 for 48 h, before transduction with lentivirus expressing OCRL1 or OCRL1 (Mut). After additional 48 h of culture, the cells were used for analysis. B , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( A ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with OCRL1 knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in C . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. D , Western blot showing ORP4L expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . The cells subjected to control (shNT), ORP4L knockdown (sh ORP4L ) alone, or combined with ORP4L rescued expression (sh ORP4L + ORP4L ) or combined with ORP4L (Mut) rescued expression (sh ORP4L + ORP4L (Mut)). Cells were transduced with lentivirus expressing shNT or sh ORP4L for 48 h, before transduction with lentivirus expressing ORP4L or ORP4L (Mut). After additional 48 h of culture, the cells were used for analysis. E , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( D ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with ORP4L knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in F . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. ∗∗∗ p < 0.001; Student's t test. CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1, OSBP, oxysterol-binding protein; ORP4L, OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PLC; phosphoinositide phospholipase C; PM, plasma membrane.
    Figure Legend Snippet: OCRL-ORP4L interaction is required for PI(4,5)P 2 consumption in the PM. A , Western blot showing OCRL expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . Cells were subjected to control (shNT), OCRL1 knockdown (sh OCRL1 ) alone, or combined with OCRL1 rescued expression (sh OCRL1 + OCRL1 ) or combined with OCRL1 (Mut) rescued expression (sh OCRL1 + OCRL1 (Mut)). Cells were transduced with lentivirus expressing shNT or sh OCRL1 for 48 h, before transduction with lentivirus expressing OCRL1 or OCRL1 (Mut). After additional 48 h of culture, the cells were used for analysis. B , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( A ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with OCRL1 knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in C . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. D , Western blot showing ORP4L expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . The cells subjected to control (shNT), ORP4L knockdown (sh ORP4L ) alone, or combined with ORP4L rescued expression (sh ORP4L + ORP4L ) or combined with ORP4L (Mut) rescued expression (sh ORP4L + ORP4L (Mut)). Cells were transduced with lentivirus expressing shNT or sh ORP4L for 48 h, before transduction with lentivirus expressing ORP4L or ORP4L (Mut). After additional 48 h of culture, the cells were used for analysis. E , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( D ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with ORP4L knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in F . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. ∗∗∗ p < 0.001; Student's t test. CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1, OSBP, oxysterol-binding protein; ORP4L, OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PLC; phosphoinositide phospholipase C; PM, plasma membrane.

    Techniques Used: Western Blot, Expressing, Transduction, Binding Assay

    pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 4 5 p 2
    ( A ) Confocal microscopy images of flat-mount P6 Inpp5k fl/fl or Inpp5k i∆EC retina stained with antibodies to PI(4,5)P 2 , PI(4)P, PI(3,4,5)P 3, PI(3,4)P 2 , or phospho-S6. Scale bar, 80 μm. Phosphoinositide staining displayed in glow-over mode; high-intensity staining is white-blue, and low-intensity staining is black-red. Graphs represent mean fluorescence intensity ± SEM of phosphoinositide or phospho-S6 relative to background retinal staining. Inpp5k fl/fl , n = 4; Inpp5k i∆EC , n = 4 [PI(4,5)P 2 ]; Inpp5k fl/fl , n = 3; Inpp5k i∆EC , n = 3 [PI(4)P]; Inpp5k fl/fl , n = 6; Inpp5k i∆EC , n = 6 [PI(3,4,5)P 3 ]; Inpp5k fl/fl , n = 5; Inpp5k i∆EC , n = 5 [PI(3,4)P 2 ]; Inpp5k fl/fl , n = 6; Inpp5k i∆EC , n = 6 (phospho-S6). Comparison between the means was assessed using Student’s t test for unpaired data. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. PI(4,5)P 2 and PI(3,4,5)P 3 signals are elevated in Inpp5k i∆EC retinal vasculature. ( B ) Schematic showing phosphoinositide interconversion mediated by INPP5K in ECs. INPP5K hydrolyzes both PI(4,5)P 2 and PI(3,4,5)P 3 . See also fig. S5.
    Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PI(4,5)P 2 -dependent regulation of endothelial tip cell specification contributes to angiogenesis"

    Article Title: PI(4,5)P 2 -dependent regulation of endothelial tip cell specification contributes to angiogenesis

    Journal: Science Advances

    doi: 10.1126/sciadv.add6911

    ( A ) Confocal microscopy images of flat-mount P6 Inpp5k fl/fl or Inpp5k i∆EC retina stained with antibodies to PI(4,5)P 2 , PI(4)P, PI(3,4,5)P 3, PI(3,4)P 2 , or phospho-S6. Scale bar, 80 μm. Phosphoinositide staining displayed in glow-over mode; high-intensity staining is white-blue, and low-intensity staining is black-red. Graphs represent mean fluorescence intensity ± SEM of phosphoinositide or phospho-S6 relative to background retinal staining. Inpp5k fl/fl , n = 4; Inpp5k i∆EC , n = 4 [PI(4,5)P 2 ]; Inpp5k fl/fl , n = 3; Inpp5k i∆EC , n = 3 [PI(4)P]; Inpp5k fl/fl , n = 6; Inpp5k i∆EC , n = 6 [PI(3,4,5)P 3 ]; Inpp5k fl/fl , n = 5; Inpp5k i∆EC , n = 5 [PI(3,4)P 2 ]; Inpp5k fl/fl , n = 6; Inpp5k i∆EC , n = 6 (phospho-S6). Comparison between the means was assessed using Student’s t test for unpaired data. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. PI(4,5)P 2 and PI(3,4,5)P 3 signals are elevated in Inpp5k i∆EC retinal vasculature. ( B ) Schematic showing phosphoinositide interconversion mediated by INPP5K in ECs. INPP5K hydrolyzes both PI(4,5)P 2 and PI(3,4,5)P 3 . See also fig. S5.
    Figure Legend Snippet: ( A ) Confocal microscopy images of flat-mount P6 Inpp5k fl/fl or Inpp5k i∆EC retina stained with antibodies to PI(4,5)P 2 , PI(4)P, PI(3,4,5)P 3, PI(3,4)P 2 , or phospho-S6. Scale bar, 80 μm. Phosphoinositide staining displayed in glow-over mode; high-intensity staining is white-blue, and low-intensity staining is black-red. Graphs represent mean fluorescence intensity ± SEM of phosphoinositide or phospho-S6 relative to background retinal staining. Inpp5k fl/fl , n = 4; Inpp5k i∆EC , n = 4 [PI(4,5)P 2 ]; Inpp5k fl/fl , n = 3; Inpp5k i∆EC , n = 3 [PI(4)P]; Inpp5k fl/fl , n = 6; Inpp5k i∆EC , n = 6 [PI(3,4,5)P 3 ]; Inpp5k fl/fl , n = 5; Inpp5k i∆EC , n = 5 [PI(3,4)P 2 ]; Inpp5k fl/fl , n = 6; Inpp5k i∆EC , n = 6 (phospho-S6). Comparison between the means was assessed using Student’s t test for unpaired data. ** P < 0.01, *** P < 0.001, and **** P < 0.0001. PI(4,5)P 2 and PI(3,4,5)P 3 signals are elevated in Inpp5k i∆EC retinal vasculature. ( B ) Schematic showing phosphoinositide interconversion mediated by INPP5K in ECs. INPP5K hydrolyzes both PI(4,5)P 2 and PI(3,4,5)P 3 . See also fig. S5.

    Techniques Used: Confocal Microscopy, Staining, Fluorescence

    ( A ) PI(4,5)P 2 or PI(4)P accumulation at the plasma membrane was assessed by indirect immunofluorescence in PIP5K1C , INPP5K , or PIP5K1C + INPP5K -siRNA HUVECs or controls grown in a confluent monolayer using antibodies to PI(4,5)P 2 or PI(4)P, respectively. Fluorescence intensity of PI(4,5)P 2 /PI(4)P at the plasma membrane relative to the cytosol in single z -plane images is expressed as mean ± SEM. More than 150 cells were analyzed per treatment over five independent transfections for PI(4,5)P 2 measurements. More than 100 cells were analyzed per treatment over three independent transfections for PI(4)P measurements. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test. ** P < 0.01 and *** P < 0.001. Scale bars, 50 μM. Increased PI(4,5)P 2 in INPP5K -siRNA HUVECs is rescued with concomitant knockdown of INPP5K and PIP5K1C . ( B ) Bright-field microscopy images of INPP5K -siRNA or PIP5K1C -siRNA HUVEC endothelial tube formation on GFR Matrigel at 16 hours in medium containing VEGF-A (50 ng/ml). Scale bars, 125 μm. Graphs depict average tubule length, tubule area, and number of interconnected tubule enclosures as means ± SEM of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. INPP5K -siRNA and PIP5K1C -siRNA HUVECs show disrupted tubule formation that is rescued with concurrent knockdown of both INPP5K and PIP5K1C . See also fig. S6.
    Figure Legend Snippet: ( A ) PI(4,5)P 2 or PI(4)P accumulation at the plasma membrane was assessed by indirect immunofluorescence in PIP5K1C , INPP5K , or PIP5K1C + INPP5K -siRNA HUVECs or controls grown in a confluent monolayer using antibodies to PI(4,5)P 2 or PI(4)P, respectively. Fluorescence intensity of PI(4,5)P 2 /PI(4)P at the plasma membrane relative to the cytosol in single z -plane images is expressed as mean ± SEM. More than 150 cells were analyzed per treatment over five independent transfections for PI(4,5)P 2 measurements. More than 100 cells were analyzed per treatment over three independent transfections for PI(4)P measurements. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test. ** P < 0.01 and *** P < 0.001. Scale bars, 50 μM. Increased PI(4,5)P 2 in INPP5K -siRNA HUVECs is rescued with concomitant knockdown of INPP5K and PIP5K1C . ( B ) Bright-field microscopy images of INPP5K -siRNA or PIP5K1C -siRNA HUVEC endothelial tube formation on GFR Matrigel at 16 hours in medium containing VEGF-A (50 ng/ml). Scale bars, 125 μm. Graphs depict average tubule length, tubule area, and number of interconnected tubule enclosures as means ± SEM of three independent experiments. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. INPP5K -siRNA and PIP5K1C -siRNA HUVECs show disrupted tubule formation that is rescued with concurrent knockdown of both INPP5K and PIP5K1C . See also fig. S6.

    Techniques Used: Immunofluorescence, Fluorescence, Transfection, Microscopy

    ( A ) Bright-field images of INPP5K -siRNA HUVEC tube formation on Matrigel for 16 hours in medium with VEGF-A (50 ng/ml) ± 2.5 μM LY294002 or ± 5 μM pan AKT inhibitor. Scale bars, 125 μm. Graphs: Interconnected tubule enclosures, tubule length, and area, as means ± SEM of three independent experiments. Statistics by ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. AKT inhibition rescues INPP5K -siRNA HUVECs tube defects. ( B ) NOTCH pathway effectors analyzed by qRT-PCR of mRNA from INPP5K -siRNA HUVECs grown on Matrigel for 16 hours with VEGF-A (50 ng/ml) ± 5 μM pan AKT inhibitor. GAPDH is endogenous control. Graph: Mean relative mRNA expression ± SEM of three independent experiments. Statistics by ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, and *** P < 0.001. Enhanced NOTCH pathway activation in INPP5K -siRNA HUVECs rescued with AKT inhibition. ( C ) INPP5K limits PI(4,5)P 2 availability for PI3K-mediated PI(3,4,5)P 3 generation and AKT activation influencing NOTCH pathway activation and EC tube formation. ( D ) Confocal images of endomucin-stained flat-mount Inpp5k fl/fl and Inpp5k i∆EC P7 retina from mice treated with 100 μg of MK2206 or DMSO vehicle, showing radial vascular expansion. Scale bars, 500 μm. Graph: Radial vascular expansion ± SEM of five retinas per genotype. Statistics by ANOVA, followed by Tukey’s post hoc test. ** P < 0.01 and **** P < 0.0001. AKT inhibition does not restore vascular expansion in Inpp5k i∆EC retina. ( E ) Confocal images of endomucin-stained flat-mount Inpp5k fl/fl and Inpp5k i∆EC P7 retina from mice treated with 100 μg of MK2206 or DMSO-vehicle. Scale bars, 50 μm. Graph: Tip cell number ± SEM. Statistics by ANOVA, followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, and *** P < 0.001. AKT inhibition rescues impaired tip cell numbers in Inpp5k i∆EC retinal vasculature. See also fig. S8.
    Figure Legend Snippet: ( A ) Bright-field images of INPP5K -siRNA HUVEC tube formation on Matrigel for 16 hours in medium with VEGF-A (50 ng/ml) ± 2.5 μM LY294002 or ± 5 μM pan AKT inhibitor. Scale bars, 125 μm. Graphs: Interconnected tubule enclosures, tubule length, and area, as means ± SEM of three independent experiments. Statistics by ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. AKT inhibition rescues INPP5K -siRNA HUVECs tube defects. ( B ) NOTCH pathway effectors analyzed by qRT-PCR of mRNA from INPP5K -siRNA HUVECs grown on Matrigel for 16 hours with VEGF-A (50 ng/ml) ± 5 μM pan AKT inhibitor. GAPDH is endogenous control. Graph: Mean relative mRNA expression ± SEM of three independent experiments. Statistics by ANOVA followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, and *** P < 0.001. Enhanced NOTCH pathway activation in INPP5K -siRNA HUVECs rescued with AKT inhibition. ( C ) INPP5K limits PI(4,5)P 2 availability for PI3K-mediated PI(3,4,5)P 3 generation and AKT activation influencing NOTCH pathway activation and EC tube formation. ( D ) Confocal images of endomucin-stained flat-mount Inpp5k fl/fl and Inpp5k i∆EC P7 retina from mice treated with 100 μg of MK2206 or DMSO vehicle, showing radial vascular expansion. Scale bars, 500 μm. Graph: Radial vascular expansion ± SEM of five retinas per genotype. Statistics by ANOVA, followed by Tukey’s post hoc test. ** P < 0.01 and **** P < 0.0001. AKT inhibition does not restore vascular expansion in Inpp5k i∆EC retina. ( E ) Confocal images of endomucin-stained flat-mount Inpp5k fl/fl and Inpp5k i∆EC P7 retina from mice treated with 100 μg of MK2206 or DMSO-vehicle. Scale bars, 50 μm. Graph: Tip cell number ± SEM. Statistics by ANOVA, followed by Tukey’s post hoc test. * P < 0.05, ** P < 0.01, and *** P < 0.001. AKT inhibition rescues impaired tip cell numbers in Inpp5k i∆EC retinal vasculature. See also fig. S8.

    Techniques Used: Inhibition, Quantitative RT-PCR, Expressing, Activation Assay, Staining

    Model depicting INPP5K regulation of PI(4,5)P 2 licenses a β-catenin/DLL4/NOTCH signaling nexus to facilitate endothelial tip cell selection.
    Figure Legend Snippet: Model depicting INPP5K regulation of PI(4,5)P 2 licenses a β-catenin/DLL4/NOTCH signaling nexus to facilitate endothelial tip cell selection.

    Techniques Used: Selection

    mouse anti pi 4 5 p 2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences mouse anti pi 4 5 p 2
    Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.
    Mouse Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture"

    Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

    Journal: STAR Protocols

    doi: 10.1016/j.xpro.2022.101549

    Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.
    Figure Legend Snippet: Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.

    Techniques Used: Immunofluorescence, Staining, Confocal Microscopy, Marker

    Immunofluorescence staining of PM PIPs and EEN1 in mouse hippocampal neurons Mouse hippocampal neurons were transfected with pAOV-CaMKIIα-mCherry-2A-3Flag on DIV5 to express mCherry as volume marker ( Note : the CaMKIIα promoter drives gene expression specifically in excitatory neurons but not inhibitory neurons) ( <xref ref-type=Kohara et al., 2020 ), fixed on DIV8 and immnunostained for PM PI3P, PI4P or PI(4,5)P 2 . DAPI-stained cell nuclei are shown in blue. (A–D) Shown are representative confocal microscopy images of PM PI3P (A), PM PI4P (B), PM PI(4,5)P 2 (C), and costaining of PM PI(4,5)P 2 and PM EEN1 (D). Lower panels are magnifications of boxed regions in the top panels. Scale bars: 5 μm." title="Immunofluorescence staining of PM PIPs and EEN1 in mouse hippocampal neurons Mouse hippocampal" property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Immunofluorescence staining of PM PIPs and EEN1 in mouse hippocampal neurons Mouse hippocampal neurons were transfected with pAOV-CaMKIIα-mCherry-2A-3Flag on DIV5 to express mCherry as volume marker ( Note : the CaMKIIα promoter drives gene expression specifically in excitatory neurons but not inhibitory neurons) ( Kohara et al., 2020 ), fixed on DIV8 and immnunostained for PM PI3P, PI4P or PI(4,5)P 2 . DAPI-stained cell nuclei are shown in blue. (A–D) Shown are representative confocal microscopy images of PM PI3P (A), PM PI4P (B), PM PI(4,5)P 2 (C), and costaining of PM PI(4,5)P 2 and PM EEN1 (D). Lower panels are magnifications of boxed regions in the top panels. Scale bars: 5 μm.

    Techniques Used: Immunofluorescence, Staining, Transfection, Marker, Expressing, Confocal Microscopy


    Figure Legend Snippet:

    Techniques Used: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting

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    Echelon Biosciences anti pi 4 5 p 2
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences anti pi 4 5 p 2 p z045
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Anti Pi 4 5 P 2 P Z045, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pi 4 5 p 2
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences anti pi 4 5 p 2 biotinylated antibody
    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.
    Anti Pi 4 5 P 2 Biotinylated Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences pi 4 5 p 2 antibody
    Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.
    Pi 4 5 P 2 Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences mouse anti pi 4 5 p 2
    Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.
    Mouse Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.

    Journal: Cell Death & Disease

    Article Title: Antipsychotics possess anti-glioblastoma activity by disrupting lysosomal function and inhibiting oncogenic signaling by stabilizing PTEN

    doi: 10.1038/s41419-024-06779-3

    Figure Lengend Snippet: A IC 50 values of phenothiazine compounds in the indicated cell lines after 48 h of treatment. B Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. C IC 50 of phenothiazines 48 h after drug treatment. D Representative clonogenic images of 200 cells 14 days post treatment with vehicle of 1.2 μM perphenazine. E Western blot analysis of empty vector (EV) and PTEN overexpression U87-MG cells treated with either vehicle or 10 µM perphenazine for 24 h. F Western blot analysis of T98G and LN18 cells treated with either vehicle or 10 µM perphenazine for 48 h. G Western blot analysis of Akt and RPS6 activation at 4 and 24 h after treatment with 10 µM perphenazine. H , I Images showing PI(3,4,5)P 3 and PI(4,5)P 2 levels in U87-MG-PTEN and U87-MG cells treated with either vehicle or 10 µM perphenazine for 48 h. J Kaplan-Meier survival curves of mice intracranially implanted with 100,000 U87-MG-PTEN cells and treated with either vehicle or 10 mg/kg perphenazine. A total of ten treatments were administered starting on day 8 post-implantation. Treatments were given for five consecutive days followed by a 2-day break for 2 weeks.

    Article Snippet: Anti-PI(4,5)P 2 (Z-P045) was purchased from Echelon Biosciences.

    Techniques: Western Blot, Plasmid Preparation, Over Expression, Activation Assay

    Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.

    Journal: The Journal of Biological Chemistry

    Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

    doi: 10.1016/j.jbc.2023.104812

    Figure Lengend Snippet: Analysis of OCRL translocation in Jurkat T-cells upon anti-CD3 stimulation. A , confocal microscopy analysis of endogenous OCRL ( green ) and ORP4L ( red ) localization in shNT- or sh ORP4L -transfected Jurkat T-cells. Cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min. Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. B , cells with wildtype OCRL1 or OCRL1 (mut) overexpression, confocal microscopy analysis of OCRL (by Xpress) and ORP4L in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml −1 ). Scale bars, 10 μm. The histograms represent the percentage of cells with OCRL translocation predominant PM localization. The percentage of cells that represent OCRL1 distribution upon anti-CD3 stimulation was quantified from >50 cells of each group. The histograms showed the results from three independent experiments. C , BiFC analysis between OCRL 1 /pVn-N1 and ORP4L /pVc-C1 or OCRL 1 (mut)/pVn-N1 and ORP4L /pVc-C1 in Jurkat T-cells. After transfected with the fragments for 24 h, cells were stimulated with or without anti-CD3 (10 μg ml −1 ) for 5 min and analyzed. Scale bars, 10 μm. The histograms showed the relative BiFC intensity from 10 cells. D , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with ORP4L knockdown. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. The histograms showed the relative OCRL protein levels in PM ( upper ) and Golgi ( lower ) from three independent experiments. E , OCRL protein levels in PM, Golgi, and total lysate of Jurkat T-cells with OCRL1 or OCRL1 (mut) overexpression. Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min. OCRL were detected by Xpress antibody. The histograms showed the relative OCRL protein levels in PM (upper) and Golgi (lower) from three independent experiments. ∗∗ p < 0.01; ∗∗∗ p < 0.001; Student's t test. BiFC, bimolecular fluorescence complementation; CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1; OSBP; oxysterol-binding protein, ORP4L; OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PM, plasma membrane.

    Article Snippet: Briefly, after centrifugation, 1 μl of suspension was spotted onto nitrocellulose membrane (Bio-Rad Laboratories), probed with PI(4,5)P 2 antibody (1:500, Echelon Biosciences), PI(4)P antibody (1:500, Echelon Biosciences), or PM internal loading control pan-cadherin antibody (Santa Cruz), and detected using an HRP-conjugated secondary antibody and enhanced chemiluminescence.

    Techniques: Translocation Assay, Confocal Microscopy, Transfection, Over Expression, Fluorescence, Binding Assay

    OCRL-ORP4L interaction is required for PI(4,5)P 2 consumption in the PM. A , Western blot showing OCRL expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . Cells were subjected to control (shNT), OCRL1 knockdown (sh OCRL1 ) alone, or combined with OCRL1 rescued expression (sh OCRL1 + OCRL1 ) or combined with OCRL1 (Mut) rescued expression (sh OCRL1 + OCRL1 (Mut)). Cells were transduced with lentivirus expressing shNT or sh OCRL1 for 48 h, before transduction with lentivirus expressing OCRL1 or OCRL1 (Mut). After additional 48 h of culture, the cells were used for analysis. B , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( A ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with OCRL1 knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in C . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. D , Western blot showing ORP4L expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . The cells subjected to control (shNT), ORP4L knockdown (sh ORP4L ) alone, or combined with ORP4L rescued expression (sh ORP4L + ORP4L ) or combined with ORP4L (Mut) rescued expression (sh ORP4L + ORP4L (Mut)). Cells were transduced with lentivirus expressing shNT or sh ORP4L for 48 h, before transduction with lentivirus expressing ORP4L or ORP4L (Mut). After additional 48 h of culture, the cells were used for analysis. E , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( D ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with ORP4L knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in F . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. ∗∗∗ p < 0.001; Student's t test. CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1, OSBP, oxysterol-binding protein; ORP4L, OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PLC; phosphoinositide phospholipase C; PM, plasma membrane.

    Journal: The Journal of Biological Chemistry

    Article Title: Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P 2 hydrolysis in the plasma membrane

    doi: 10.1016/j.jbc.2023.104812

    Figure Lengend Snippet: OCRL-ORP4L interaction is required for PI(4,5)P 2 consumption in the PM. A , Western blot showing OCRL expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . Cells were subjected to control (shNT), OCRL1 knockdown (sh OCRL1 ) alone, or combined with OCRL1 rescued expression (sh OCRL1 + OCRL1 ) or combined with OCRL1 (Mut) rescued expression (sh OCRL1 + OCRL1 (Mut)). Cells were transduced with lentivirus expressing shNT or sh OCRL1 for 48 h, before transduction with lentivirus expressing OCRL1 or OCRL1 (Mut). After additional 48 h of culture, the cells were used for analysis. B , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( A ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with OCRL1 knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in C . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. D , Western blot showing ORP4L expression in Jurkat T-cells expressing PI(4,5)P 2 probe GFP-PH PLCδ1 . The cells subjected to control (shNT), ORP4L knockdown (sh ORP4L ) alone, or combined with ORP4L rescued expression (sh ORP4L + ORP4L ) or combined with ORP4L (Mut) rescued expression (sh ORP4L + ORP4L (Mut)). Cells were transduced with lentivirus expressing shNT or sh ORP4L for 48 h, before transduction with lentivirus expressing ORP4L or ORP4L (Mut). After additional 48 h of culture, the cells were used for analysis. E , PI(4,5)P 2 levels at the PM as detected by PI(4,5)P 2 probe GFP-PH PLCδ1 in Jurkat T-cells treated as panel ( D ). Cells were stimulated with 10 μg ml −1 anti-CD3 for 5 min before analyzed. The cells with ORP4L knockdown or rescue expression were considered for analysis, and the relative PI(4,5)P 2 levels expressed as fold change are shown in F . The data represent mean ± SD (n = 10 cells). Scale bar, 10 μm. ∗∗∗ p < 0.001; Student's t test. CD3, cluster of differentiation 3; OCRL, oculocerebrorenal syndrome of Lowe 1, OSBP, oxysterol-binding protein; ORP4L, OSBP-related protein 4L; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PLC; phosphoinositide phospholipase C; PM, plasma membrane.

    Article Snippet: Briefly, after centrifugation, 1 μl of suspension was spotted onto nitrocellulose membrane (Bio-Rad Laboratories), probed with PI(4,5)P 2 antibody (1:500, Echelon Biosciences), PI(4)P antibody (1:500, Echelon Biosciences), or PM internal loading control pan-cadherin antibody (Santa Cruz), and detected using an HRP-conjugated secondary antibody and enhanced chemiluminescence.

    Techniques: Western Blot, Expressing, Transduction, Binding Assay

    Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.

    Journal: STAR Protocols

    Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

    doi: 10.1016/j.xpro.2022.101549

    Figure Lengend Snippet: Immunofluorescence staining of intracellular PIPs and Rab8/TGN46 in mouse hippocampal neurons Mouse hippocampal neurons were fixed on DIV12 and co-immnunostained for intracellular PI3P, PI4P or PI(4,5)P 2 and Rab8 or TGN46. DAPI-stained cell nuclei are shown in blue. (A–F) Shown are representative confocal microscopy images of intracellular PI3P and Rab8 (A), intracellular PI4P and Rab8 (B), intracellular PI(4,5)P 2 and Rab8 (C), intracellular PI3P and TGN46 (D), intracellular PI4P and TGN46 (E), intracellular PI(4,5)P 2 and TGN46 (F). Rab8: protein marker for secretory endosomes, TGN46: protein marker for the trans -Golgi network. Lower panels are magnifications of outlined regions in the top panels. Scale bars: 5 μm.

    Article Snippet: Mouse anti-PI(4,5)P 2 , IgM (1:50 - 1:150) , Echelon Biosciences , Cat# Z-A045, RRID: AB_427211.

    Techniques: Immunofluorescence, Staining, Confocal Microscopy, Marker

    Immunofluorescence staining of PM PIPs and EEN1 in mouse hippocampal neurons Mouse hippocampal neurons were transfected with pAOV-CaMKIIα-mCherry-2A-3Flag on DIV5 to express mCherry as volume marker ( Note : the CaMKIIα promoter drives gene expression specifically in excitatory neurons but not inhibitory neurons) ( <xref ref-type=Kohara et al., 2020 ), fixed on DIV8 and immnunostained for PM PI3P, PI4P or PI(4,5)P 2 . DAPI-stained cell nuclei are shown in blue. (A–D) Shown are representative confocal microscopy images of PM PI3P (A), PM PI4P (B), PM PI(4,5)P 2 (C), and costaining of PM PI(4,5)P 2 and PM EEN1 (D). Lower panels are magnifications of boxed regions in the top panels. Scale bars: 5 μm." width="100%" height="100%">

    Journal: STAR Protocols

    Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

    doi: 10.1016/j.xpro.2022.101549

    Figure Lengend Snippet: Immunofluorescence staining of PM PIPs and EEN1 in mouse hippocampal neurons Mouse hippocampal neurons were transfected with pAOV-CaMKIIα-mCherry-2A-3Flag on DIV5 to express mCherry as volume marker ( Note : the CaMKIIα promoter drives gene expression specifically in excitatory neurons but not inhibitory neurons) ( Kohara et al., 2020 ), fixed on DIV8 and immnunostained for PM PI3P, PI4P or PI(4,5)P 2 . DAPI-stained cell nuclei are shown in blue. (A–D) Shown are representative confocal microscopy images of PM PI3P (A), PM PI4P (B), PM PI(4,5)P 2 (C), and costaining of PM PI(4,5)P 2 and PM EEN1 (D). Lower panels are magnifications of boxed regions in the top panels. Scale bars: 5 μm.

    Article Snippet: Mouse anti-PI(4,5)P 2 , IgM (1:50 - 1:150) , Echelon Biosciences , Cat# Z-A045, RRID: AB_427211.

    Techniques: Immunofluorescence, Staining, Transfection, Marker, Expressing, Confocal Microscopy

    Journal: STAR Protocols

    Article Title: Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

    doi: 10.1016/j.xpro.2022.101549

    Figure Lengend Snippet:

    Article Snippet: Mouse anti-PI(4,5)P 2 , IgM (1:50 - 1:150) , Echelon Biosciences , Cat# Z-A045, RRID: AB_427211.

    Techniques: Recombinant, Electron Microscopy, Software, Microscopy, Cell Culture, In Vitro, Cell Counting