phosphatidylinositol dic8  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol dic8
    Phosphatidylinositol Dic8, supplied by Echelon Biosciences, 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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    synthetic lipids dic8 pi p 0008  (Echelon Biosciences)


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    Echelon Biosciences synthetic lipids dic8 pi p 0008
    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Synthetic Lipids Dic8 Pi P 0008, 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 "Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer"

    Article Title: Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

    Journal: The EMBO Journal

    doi: 10.1038/s44318-024-00085-6

    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Figure Legend Snippet: ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Techniques Used: Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy

    synthetic lipids dic8 pi p 0008  (Echelon Biosciences)


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    Echelon Biosciences synthetic lipids dic8 pi p 0008
    Synthetic Lipids Dic8 Pi P 0008, 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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    phosphatidylinositol dic8  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol dic8
    Phosphatidylinositol Dic8, supplied by Echelon Biosciences, 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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    dic8 pip 2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pip 2
    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), <t>diC8-PIP</t> 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).
    Dic8 Pip 2, supplied by Echelon Biosciences, 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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    1) Product Images from "Hyperforin/HP- β -Cyclodextrin Enhances Mechanosensitive Ca 2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing"

    Article Title: Hyperforin/HP- β -Cyclodextrin Enhances Mechanosensitive Ca 2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing

    Journal: BioMed Research International

    doi: 10.1155/2017/8701801

    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), diC8-PIP 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).
    Figure Legend Snippet: The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), diC8-PIP 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).

    Techniques Used: Fluorescence, Blocking Assay

    phosphatidylinositol  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol
    Phosphatidylinositol, supplied by Echelon Biosciences, 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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    water soluble dic8 pip3  (Echelon Biosciences)


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    Echelon Biosciences water soluble dic8 pip3
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    phosphatidylinositol  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol
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    dic8 pip 2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pip 2
    SUMO1 decreases the effect of <t>PIP</t> <t>2</t> on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also <xref ref-type=Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1. " width="250" height="auto" />
    Dic8 Pip 2, supplied by Echelon Biosciences, 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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    1) Product Images from "Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2"

    Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

    Journal: iScience

    doi: 10.1016/j.isci.2022.104969

    SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also <xref ref-type=Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1. " title="SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1.

    Techniques Used: Injection, Expressing, Two Tailed Test, Inhibition, Activation Assay

    SUMOylation interferes with the activation of Kir2.1 channels by diC8-PIP 2 Kir2.1 or Kir2.1-K49R channels were expressed in Xenopus oocytes with or without Ubc9 and SUMO1 and the currents from excised macropatches were studied by patch-clamp recording at −100mV. See the for experimental details. Data are mean ± s.d. for 7-9 experiments per condition. ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (A) Example data showing that Kir2.1 (blue, upper ) or Kir2.1-K49R (orange, lower ) channels were first exposed to poly-L-lysine (PLL; 20 μg/mL, not shown) and then to increasing concentrations of diC8-PIP 2 , as indicated. Currents were then deactivated by re-exposure to PLL. Patches were studied with multiple concentrations of diC8-PIP 2 always including 10 μM as a reference. (B) Mean current magnitudes plotted against the concentration of diC8-PIP 2 . (C) The data are normalized to obtain EC 50 values. (D) Summary data show that diC8-PIP 2 activates Kir2.1 with a mean EC 50 of 3.3 ± 1 μM. The EC 50 increases to 9.8 ± 2 μM when patches were excised from oocytes overexpressing SUMO1 and Ubc9. DiC8-PIP 2 activated Kir2.1-K49R with an EC 50 of 2.5 ± 1 μM with or without co-expression of SUMO1.
    Figure Legend Snippet: SUMOylation interferes with the activation of Kir2.1 channels by diC8-PIP 2 Kir2.1 or Kir2.1-K49R channels were expressed in Xenopus oocytes with or without Ubc9 and SUMO1 and the currents from excised macropatches were studied by patch-clamp recording at −100mV. See the for experimental details. Data are mean ± s.d. for 7-9 experiments per condition. ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (A) Example data showing that Kir2.1 (blue, upper ) or Kir2.1-K49R (orange, lower ) channels were first exposed to poly-L-lysine (PLL; 20 μg/mL, not shown) and then to increasing concentrations of diC8-PIP 2 , as indicated. Currents were then deactivated by re-exposure to PLL. Patches were studied with multiple concentrations of diC8-PIP 2 always including 10 μM as a reference. (B) Mean current magnitudes plotted against the concentration of diC8-PIP 2 . (C) The data are normalized to obtain EC 50 values. (D) Summary data show that diC8-PIP 2 activates Kir2.1 with a mean EC 50 of 3.3 ± 1 μM. The EC 50 increases to 9.8 ± 2 μM when patches were excised from oocytes overexpressing SUMO1 and Ubc9. DiC8-PIP 2 activated Kir2.1-K49R with an EC 50 of 2.5 ± 1 μM with or without co-expression of SUMO1.

    Techniques Used: Activation Assay, Patch Clamp, Two Tailed Test, Concentration Assay, Expressing

    PIP 2 opposed hypoxic inhibition of I K1 I K1 was studied in rat ventricular cardiomyocytes (RVCMs) using a whole-cell patch-clamp recording. To the knockdown Kir2.1 expression, RVCMs were transduced with lentiviral particles carrying eGFP and shRNA targeting KCNJ2. Kir2.1 knockdown cells (Kir2.1 kd -CMs) were identified by the expression of eGFP. Cells were studied with a control pipette solution (blue), or with pipette solutions containing purified SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Where indicated, the control pipette solution contained diC8 PIP 2 . Paired patch-clamp data were analyzed by Students t -test; ∗∗∗, p < 0.01. The proximity ligation assay (PLA) for native Kir2.1-SUMO1 interactions was performed as described in the and analyzed using an unpaired Mann-Whitney rank test, ∗∗∗∗p < 0.001. See also <xref ref-type=Figures S7 and . (A) Left , Example sweeps show that I K1 is diminished in Kir2.1 kd -CMs and the remaining current is insensitive to acute hypoxia. Kir2.1 kd -CMs are identified by expression of GFP ( inset , scale bar = 10 μm). Right , summary data from 8 to 10 cells per group show that the regulation of I K1 by hypoxia, SUMO1, and SENP1 is lost in Kir2.1 kd -CMs. (B) The magnitude and the regulation of I K1 by hypoxia, SUMO1, and SENP1 are unaltered when cells are treated with a control, scrambled shRNA; 7-8 cells per group. (C) Left , Example traces to show that including diC8-PIP 2 in the pipette solution reduces the hypoxic inhibition of I K1 in RVCMs in a concentration-dependent manner. The arrow indicates the change in current magnitude between exposure to ambient O 2 and 2% O 2 in the same cell. Right , Concentration-response curve showing that including diC8-PIP 2 in the pipette opposes hypoxic inhibition of I K1 in control RVCMs and RVCMs expressing the scrambled shRNA. The hypoxic-response is diminished in Kir2.1 kd -CMs. Data are mean ± s.d. for 7-8 cells per condition. (D) DiC8-PIP 2 decreases hypoxic inhibition of Kir2.1 channels expressed in HEK293T cells (control) in a concentration-dependent manner using the experimental paradigm described in C, above. Current inhibition is diminished when SENP1 is included in the recording pipette and is not observed when Kir2.1-K49Q channels are studied. Data are mean ± s.d. for 6 cells per condition. (E) PLA shows that native Kir2.1 colocalizes with SUMO1 in RVCMs and that Kir2.1-SUMO interactions are increased by acute hypoxia. Kir2.2 and Kir2.3 do not associate with SUMO1. Representative data are shown with DAPI-labeled nuclei in red and PLA interactions in green for ease of visualization. Summary data are obtained from multiple experiments each with multiple fields of view containing 20-30 nuclei. The scale bar is 10 μm. " title="PIP 2 opposed hypoxic inhibition of I K1 I ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: PIP 2 opposed hypoxic inhibition of I K1 I K1 was studied in rat ventricular cardiomyocytes (RVCMs) using a whole-cell patch-clamp recording. To the knockdown Kir2.1 expression, RVCMs were transduced with lentiviral particles carrying eGFP and shRNA targeting KCNJ2. Kir2.1 knockdown cells (Kir2.1 kd -CMs) were identified by the expression of eGFP. Cells were studied with a control pipette solution (blue), or with pipette solutions containing purified SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Where indicated, the control pipette solution contained diC8 PIP 2 . Paired patch-clamp data were analyzed by Students t -test; ∗∗∗, p < 0.01. The proximity ligation assay (PLA) for native Kir2.1-SUMO1 interactions was performed as described in the and analyzed using an unpaired Mann-Whitney rank test, ∗∗∗∗p < 0.001. See also Figures S7 and . (A) Left , Example sweeps show that I K1 is diminished in Kir2.1 kd -CMs and the remaining current is insensitive to acute hypoxia. Kir2.1 kd -CMs are identified by expression of GFP ( inset , scale bar = 10 μm). Right , summary data from 8 to 10 cells per group show that the regulation of I K1 by hypoxia, SUMO1, and SENP1 is lost in Kir2.1 kd -CMs. (B) The magnitude and the regulation of I K1 by hypoxia, SUMO1, and SENP1 are unaltered when cells are treated with a control, scrambled shRNA; 7-8 cells per group. (C) Left , Example traces to show that including diC8-PIP 2 in the pipette solution reduces the hypoxic inhibition of I K1 in RVCMs in a concentration-dependent manner. The arrow indicates the change in current magnitude between exposure to ambient O 2 and 2% O 2 in the same cell. Right , Concentration-response curve showing that including diC8-PIP 2 in the pipette opposes hypoxic inhibition of I K1 in control RVCMs and RVCMs expressing the scrambled shRNA. The hypoxic-response is diminished in Kir2.1 kd -CMs. Data are mean ± s.d. for 7-8 cells per condition. (D) DiC8-PIP 2 decreases hypoxic inhibition of Kir2.1 channels expressed in HEK293T cells (control) in a concentration-dependent manner using the experimental paradigm described in C, above. Current inhibition is diminished when SENP1 is included in the recording pipette and is not observed when Kir2.1-K49Q channels are studied. Data are mean ± s.d. for 6 cells per condition. (E) PLA shows that native Kir2.1 colocalizes with SUMO1 in RVCMs and that Kir2.1-SUMO interactions are increased by acute hypoxia. Kir2.2 and Kir2.3 do not associate with SUMO1. Representative data are shown with DAPI-labeled nuclei in red and PLA interactions in green for ease of visualization. Summary data are obtained from multiple experiments each with multiple fields of view containing 20-30 nuclei. The scale bar is 10 μm.

    Techniques Used: Inhibition, Patch Clamp, Expressing, Transduction, shRNA, Transferring, Purification, Proximity Ligation Assay, MANN-WHITNEY, Concentration Assay, Labeling


    Figure Legend Snippet:

    Techniques Used: Recombinant, shRNA, In Situ, Software, Microscopy

    dic8 pip 2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pip 2
    SUMO1 decreases the effect of <t>PIP</t> <t>2</t> on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also <xref ref-type=Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1. " width="250" height="auto" />
    Dic8 Pip 2, supplied by Echelon Biosciences, 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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    1) Product Images from "Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2"

    Article Title: Hypoxia inhibits the cardiac I K1 current through SUMO targeting Kir2.1 activation by PIP 2

    Journal: iScience

    doi: 10.1016/j.isci.2022.104969

    SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also <xref ref-type=Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1. " title="SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: SUMO1 decreases the effect of PIP 2 on Kir2.1 Rat Kir2.1 channel currents were studied in Xenopus oocytes by two-electrode voltage-clamp. Currents were evoked in a high external K + recording buffer and measured at −80 mV, as described in the . To study the effects of SUMOylation and deSUMOylation, the oocytes were also injected with cRNAs expressing Ubc9 and SUMO1 (orange) or SENP1 (magenta) and compared to control oocytes (blue). Endogenous PIP 2 was dephosphorylated at the 5′ position by co-expressing CIBN-CAAX and CRY2-5′ptase OCRL .(5′ptase) and activating this optogenetic system with a 460 nm LED were indicated by the cyan box. Data are mean ± s.d. for 6-8 oocytes per group, ∗p < 0 . 05 ,∗∗p < 0 . 01 , ∗∗∗p < 0 . 001 , ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test; ∗p < 0 . 05 , unpaired, two-tailed Student’s t test. See also Figures S3–S6 . (A) Example time courses show that the rate of inhibition of Kir2.1 by the activation of CRY-5′ptase is increased by SUMO1 and slowed by SENP1. (B) The tau of inhibition is determined by fitting the data with a mono-exponential function. (C) Left , Summary data showing the effect of co-expressed SUMO1 or SENP1 on the peak Kir2.1 current before and after the activation of 5′ptase as well as the percentage of the current remaining ( middle ) and the tau of inhibition ( right ). (D) Left , Summary data showing that co-expression of SUMO1 or SENP1 does not alter peak Kir2.1-K49Q current before and after the activation of 5′ptase. The percentage of Kir2.1-K49Q current remaining ( middle ) and the tau of inhibition ( right ) are insensitive to SUMO1 or SENP1.

    Techniques Used: Injection, Expressing, Two Tailed Test, Inhibition, Activation Assay

    SUMOylation interferes with the activation of Kir2.1 channels by diC8-PIP 2 Kir2.1 or Kir2.1-K49R channels were expressed in Xenopus oocytes with or without Ubc9 and SUMO1 and the currents from excised macropatches were studied by patch-clamp recording at −100mV. See the for experimental details. Data are mean ± s.d. for 7-9 experiments per condition. ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (A) Example data showing that Kir2.1 (blue, upper ) or Kir2.1-K49R (orange, lower ) channels were first exposed to poly-L-lysine (PLL; 20 μg/mL, not shown) and then to increasing concentrations of diC8-PIP 2 , as indicated. Currents were then deactivated by re-exposure to PLL. Patches were studied with multiple concentrations of diC8-PIP 2 always including 10 μM as a reference. (B) Mean current magnitudes plotted against the concentration of diC8-PIP 2 . (C) The data are normalized to obtain EC 50 values. (D) Summary data show that diC8-PIP 2 activates Kir2.1 with a mean EC 50 of 3.3 ± 1 μM. The EC 50 increases to 9.8 ± 2 μM when patches were excised from oocytes overexpressing SUMO1 and Ubc9. DiC8-PIP 2 activated Kir2.1-K49R with an EC 50 of 2.5 ± 1 μM with or without co-expression of SUMO1.
    Figure Legend Snippet: SUMOylation interferes with the activation of Kir2.1 channels by diC8-PIP 2 Kir2.1 or Kir2.1-K49R channels were expressed in Xenopus oocytes with or without Ubc9 and SUMO1 and the currents from excised macropatches were studied by patch-clamp recording at −100mV. See the for experimental details. Data are mean ± s.d. for 7-9 experiments per condition. ∗∗∗∗p < 0 . 0001 , paired, two-tailed Student’s t test. (A) Example data showing that Kir2.1 (blue, upper ) or Kir2.1-K49R (orange, lower ) channels were first exposed to poly-L-lysine (PLL; 20 μg/mL, not shown) and then to increasing concentrations of diC8-PIP 2 , as indicated. Currents were then deactivated by re-exposure to PLL. Patches were studied with multiple concentrations of diC8-PIP 2 always including 10 μM as a reference. (B) Mean current magnitudes plotted against the concentration of diC8-PIP 2 . (C) The data are normalized to obtain EC 50 values. (D) Summary data show that diC8-PIP 2 activates Kir2.1 with a mean EC 50 of 3.3 ± 1 μM. The EC 50 increases to 9.8 ± 2 μM when patches were excised from oocytes overexpressing SUMO1 and Ubc9. DiC8-PIP 2 activated Kir2.1-K49R with an EC 50 of 2.5 ± 1 μM with or without co-expression of SUMO1.

    Techniques Used: Activation Assay, Patch Clamp, Two Tailed Test, Concentration Assay, Expressing

    PIP 2 opposed hypoxic inhibition of I K1 I K1 was studied in rat ventricular cardiomyocytes (RVCMs) using a whole-cell patch-clamp recording. To the knockdown Kir2.1 expression, RVCMs were transduced with lentiviral particles carrying eGFP and shRNA targeting KCNJ2. Kir2.1 knockdown cells (Kir2.1 kd -CMs) were identified by the expression of eGFP. Cells were studied with a control pipette solution (blue), or with pipette solutions containing purified SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Where indicated, the control pipette solution contained diC8 PIP 2 . Paired patch-clamp data were analyzed by Students t -test; ∗∗∗, p < 0.01. The proximity ligation assay (PLA) for native Kir2.1-SUMO1 interactions was performed as described in the and analyzed using an unpaired Mann-Whitney rank test, ∗∗∗∗p < 0.001. See also <xref ref-type=Figures S7 and . (A) Left , Example sweeps show that I K1 is diminished in Kir2.1 kd -CMs and the remaining current is insensitive to acute hypoxia. Kir2.1 kd -CMs are identified by expression of GFP ( inset , scale bar = 10 μm). Right , summary data from 8 to 10 cells per group show that the regulation of I K1 by hypoxia, SUMO1, and SENP1 is lost in Kir2.1 kd -CMs. (B) The magnitude and the regulation of I K1 by hypoxia, SUMO1, and SENP1 are unaltered when cells are treated with a control, scrambled shRNA; 7-8 cells per group. (C) Left , Example traces to show that including diC8-PIP 2 in the pipette solution reduces the hypoxic inhibition of I K1 in RVCMs in a concentration-dependent manner. The arrow indicates the change in current magnitude between exposure to ambient O 2 and 2% O 2 in the same cell. Right , Concentration-response curve showing that including diC8-PIP 2 in the pipette opposes hypoxic inhibition of I K1 in control RVCMs and RVCMs expressing the scrambled shRNA. The hypoxic-response is diminished in Kir2.1 kd -CMs. Data are mean ± s.d. for 7-8 cells per condition. (D) DiC8-PIP 2 decreases hypoxic inhibition of Kir2.1 channels expressed in HEK293T cells (control) in a concentration-dependent manner using the experimental paradigm described in C, above. Current inhibition is diminished when SENP1 is included in the recording pipette and is not observed when Kir2.1-K49Q channels are studied. Data are mean ± s.d. for 6 cells per condition. (E) PLA shows that native Kir2.1 colocalizes with SUMO1 in RVCMs and that Kir2.1-SUMO interactions are increased by acute hypoxia. Kir2.2 and Kir2.3 do not associate with SUMO1. Representative data are shown with DAPI-labeled nuclei in red and PLA interactions in green for ease of visualization. Summary data are obtained from multiple experiments each with multiple fields of view containing 20-30 nuclei. The scale bar is 10 μm. " title="PIP 2 opposed hypoxic inhibition of I K1 I ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: PIP 2 opposed hypoxic inhibition of I K1 I K1 was studied in rat ventricular cardiomyocytes (RVCMs) using a whole-cell patch-clamp recording. To the knockdown Kir2.1 expression, RVCMs were transduced with lentiviral particles carrying eGFP and shRNA targeting KCNJ2. Kir2.1 knockdown cells (Kir2.1 kd -CMs) were identified by the expression of eGFP. Cells were studied with a control pipette solution (blue), or with pipette solutions containing purified SUMO1 (1 μM, orange) or SENP1 (2 μM, magenta). Where indicated, the control pipette solution contained diC8 PIP 2 . Paired patch-clamp data were analyzed by Students t -test; ∗∗∗, p < 0.01. The proximity ligation assay (PLA) for native Kir2.1-SUMO1 interactions was performed as described in the and analyzed using an unpaired Mann-Whitney rank test, ∗∗∗∗p < 0.001. See also Figures S7 and . (A) Left , Example sweeps show that I K1 is diminished in Kir2.1 kd -CMs and the remaining current is insensitive to acute hypoxia. Kir2.1 kd -CMs are identified by expression of GFP ( inset , scale bar = 10 μm). Right , summary data from 8 to 10 cells per group show that the regulation of I K1 by hypoxia, SUMO1, and SENP1 is lost in Kir2.1 kd -CMs. (B) The magnitude and the regulation of I K1 by hypoxia, SUMO1, and SENP1 are unaltered when cells are treated with a control, scrambled shRNA; 7-8 cells per group. (C) Left , Example traces to show that including diC8-PIP 2 in the pipette solution reduces the hypoxic inhibition of I K1 in RVCMs in a concentration-dependent manner. The arrow indicates the change in current magnitude between exposure to ambient O 2 and 2% O 2 in the same cell. Right , Concentration-response curve showing that including diC8-PIP 2 in the pipette opposes hypoxic inhibition of I K1 in control RVCMs and RVCMs expressing the scrambled shRNA. The hypoxic-response is diminished in Kir2.1 kd -CMs. Data are mean ± s.d. for 7-8 cells per condition. (D) DiC8-PIP 2 decreases hypoxic inhibition of Kir2.1 channels expressed in HEK293T cells (control) in a concentration-dependent manner using the experimental paradigm described in C, above. Current inhibition is diminished when SENP1 is included in the recording pipette and is not observed when Kir2.1-K49Q channels are studied. Data are mean ± s.d. for 6 cells per condition. (E) PLA shows that native Kir2.1 colocalizes with SUMO1 in RVCMs and that Kir2.1-SUMO interactions are increased by acute hypoxia. Kir2.2 and Kir2.3 do not associate with SUMO1. Representative data are shown with DAPI-labeled nuclei in red and PLA interactions in green for ease of visualization. Summary data are obtained from multiple experiments each with multiple fields of view containing 20-30 nuclei. The scale bar is 10 μm.

    Techniques Used: Inhibition, Patch Clamp, Expressing, Transduction, shRNA, Transferring, Purification, Proximity Ligation Assay, MANN-WHITNEY, Concentration Assay, Labeling


    Figure Legend Snippet:

    Techniques Used: Recombinant, shRNA, In Situ, Software, Microscopy

    dic8 pip 3  (Echelon Biosciences)


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    Echelon Biosciences dic8 pip 3
    TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a <t>diC8-PI(4,5)P</t> 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , <t>diC8-PI(4,5)P</t> 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of <t>diC8-PI(4,5)P</t> 2 with Ca 2+ (N = 8). <t>diC8-PI(4,5)P</t> 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
    Dic8 Pip 3, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction"

    Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

    Journal: The Journal of Biological Chemistry

    doi: 10.1016/j.jbc.2022.102264

    TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a diC8-PI(4,5)P 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , diC8-PI(4,5)P 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 with Ca 2+ (N = 8). diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
    Figure Legend Snippet: TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a diC8-PI(4,5)P 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , diC8-PI(4,5)P 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 with Ca 2+ (N = 8). diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.

    Techniques Used: Stable Transfection

    Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP 3 (diC8-PIP 3 ), PI(3,4)P 2 (diC8-PI(3,4)P 2 ), and PI(3,5)P 2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PIP 3 (N = 7), diC8-PI(3,4)P 2 (N = 6), or diC8-PI(3,5)P 2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP 3 ( B ), diC8-PI(3,4)P 2 ( C ), or diC8-PI(3,5)P 2 ( D ). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P 2 , dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP 3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP 3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.
    Figure Legend Snippet: Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP 3 (diC8-PIP 3 ), PI(3,4)P 2 (diC8-PI(3,4)P 2 ), and PI(3,5)P 2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PIP 3 (N = 7), diC8-PI(3,4)P 2 (N = 6), or diC8-PI(3,5)P 2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP 3 ( B ), diC8-PI(3,4)P 2 ( C ), or diC8-PI(3,5)P 2 ( D ). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P 2 , dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP 3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP 3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.

    Techniques Used: Stable Transfection

    Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P ( A ), 100 μM diC8-PI5P ( B ), or 100 μM diC8-PI4P ( D ). C , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P 2 , as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.
    Figure Legend Snippet: Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P ( A ), 100 μM diC8-PI5P ( B ), or 100 μM diC8-PI4P ( D ). C , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P 2 , as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.

    Techniques Used: Stable Transfection

    Docking suggests key PI(4,5)P 2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P 2 or IP 3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A , position of diC8-PI(4,5)P 2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB ). B , detailed view of the hypothesized PI(4,5)P 2 –xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C , superposition of docked IP 3 (foreground) on the PI(4,5)P 2 –xTMEM16A (transparency) interaction. diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P 2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.
    Figure Legend Snippet: Docking suggests key PI(4,5)P 2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P 2 or IP 3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A , position of diC8-PI(4,5)P 2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB ). B , detailed view of the hypothesized PI(4,5)P 2 –xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C , superposition of docked IP 3 (foreground) on the PI(4,5)P 2 –xTMEM16A (transparency) interaction. diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P 2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.

    Techniques Used:

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    Echelon Biosciences phosphatidylinositol dic8
    Phosphatidylinositol Dic8, supplied by Echelon Biosciences, 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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    Echelon Biosciences synthetic lipids dic8 pi p 0008
    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
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    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), <t>diC8-PIP</t> 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).
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    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), <t>diC8-PIP</t> 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).
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    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), <t>diC8-PIP</t> 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).
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    TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a <t>diC8-PI(4,5)P</t> 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , <t>diC8-PI(4,5)P</t> 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of <t>diC8-PI(4,5)P</t> 2 with Ca 2+ (N = 8). <t>diC8-PI(4,5)P</t> 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.
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    Image Search Results


    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Journal: The EMBO Journal

    Article Title: Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

    doi: 10.1038/s44318-024-00085-6

    Figure Lengend Snippet: ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Article Snippet: Synthetic lipids diC8 PI (P-0008), diC8 PI(3)P (P-3008), diC8 PI(4)P (P-4008), diC8 PI(5)P (P-5008), diC8 PI(3,4)P 2 (P-3408), diC8 PI(3,5)P 2 (P-3508), diC8 PI(4,5)P 2 (P-4508), diC8 PI(3,4,5)P 3 (P-3908), diC16 PI(4,5)P 2 (P-4516), and diC16 PI(3,4,5)P 3 (P-3916) were purchased from Echelon Bioscience.

    Techniques: Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy

    The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), diC8-PIP 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).

    Journal: BioMed Research International

    Article Title: Hyperforin/HP- β -Cyclodextrin Enhances Mechanosensitive Ca 2+ Signaling in HaCaT Keratinocytes and in Atopic Skin Ex Vivo Which Accelerates Wound Healing

    doi: 10.1155/2017/8701801

    Figure Lengend Snippet: The effects of various inhibitors on the stretch-induced Ca 2+ responses and wound closure. (a) The time course of changes in the fluorescence intensity of Fluo-8 due to a transient 20% stretch in hyperforin/HP- β -CD-treated HaCaT cells (control). Each color trace indicated the data at different distances of 0, 60, 120, 180, and 240 from the wound edge (inset image). The intensity was normalized to the peak value obtained with ionomycin treatment at the end of each experiment. (b) The effects of Gd 3+ on the stretch-induced Ca 2+ response as a typical example of the blocking effects of the inhibitors. Gd 3+ (10 μ M) was applied at 10 min before the application of a 20% stretch. (c) The effects of various inhibitors on 20% transient stretch-induced Ca 2+ responses in hyperforin/HP- β -CD-treated HaCaT cells. The intensity traces at each distance from the wound edge were averaged and normalized to the peak intensity obtained with ionomycin treatment. The data show the average of the peak values obtained in 3–6 separate experiments. Various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), U73122 (10 μ M), diC8-PIP 2 (5 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M), were applied at 10 min before the stretch stimulation. A Ca 2+ -free condition was achieved by changing the medium to Ca 2+ -free medium that contained 0.5 μ M EGTA. All of the quantitative data are shown as the mean (±SEM). (d) The effects of various inhibitors on the stretch facilitated wound closure in hyperforin/HP- β -CD-treated HaCaT cells. Confluent cell cultures were scratched and allowed to migrate for 6 h under a sustained 20% stretch in a medium that contained various inhibitors, including CBX (100 μ M), apyrase (20 Unit/mL), suramin (100 μ M), Gd 3+ (10 μ M), and GsMTx-4 (5 μ M) or in nominally Ca 2+ -free medium. shTRPC6 was applied to the cells for 3 h; the cells were then grown to confluence. Representative DIC images (upper panel) and the means of 3–8 wound closure experiments at 6 h after scratching (lower panel) are shown. All of the quantitative data are shown as the mean (±SEM).

    Article Snippet: The other chemicals and reagents were as follows: carbenoxolone disodium salt (CBX), apyrase (from potato), GdCl 3 , U73122, and Cremophor EL (Sigma-Aldrich, St. Louis, MO); ionomycin (Calbiochem, San Diego, CA); suramin hexasodium (RBI, Natick, MA); GsMTx-4 (Peptide Institute, Osaka, Japan); diC8-PIP 2 (Echelon Biosciences, Salt Lake City, UT); dispase (Godo Shusei, Tokyo, Japan); Fluo-8 AM (AAT Bioquest, Sunnyvale, CA); Cellmatrix type IA (Nitta Gelatin, Osaka, Japan); Lipofectamine (18324, Invitrogen, Carlsbad, CA); DME/F12 (D9785; Sigma-Aldrich, St. Louis, MO); FBS (12483; Gibco, Carlsbad, CA).

    Techniques: Fluorescence, Blocking Assay

    TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a diC8-PI(4,5)P 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , diC8-PI(4,5)P 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 with Ca 2+ (N = 8). diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

    doi: 10.1016/j.jbc.2022.102264

    Figure Lengend Snippet: TMEM16A Ca 2+ -evoked Cl − currents rundown in excised patches and are recovered by a diC8-PI(4,5)P 2 application. A , example currents recorded at the indicated times during 150 ms steps to −60 and +60 mV, recorded using excised inside–out macropatches from Xenopus laevis oocytes. B , normalized plot of current measured at −60 mV versus time, fit with a single exponential ( red line ). C , box plot distribution of the rate of current decay (τ), measured by fitting plots of relative current versus time with single exponentials (N = 11). The central line denotes the median, the box denotes 25 to 75% of the data, and the whiskers represent 10 to 90% of the data. D , a soluble synthetic analog of PI(4,5)P 2 , diC8-PI(4,5)P 2 , was applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. E , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 with Ca 2+ (N = 8). diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A.

    Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

    Techniques: Stable Transfection

    Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP 3 (diC8-PIP 3 ), PI(3,4)P 2 (diC8-PI(3,4)P 2 ), and PI(3,5)P 2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PIP 3 (N = 7), diC8-PI(3,4)P 2 (N = 6), or diC8-PI(3,5)P 2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP 3 ( B ), diC8-PI(3,4)P 2 ( C ), or diC8-PI(3,5)P 2 ( D ). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P 2 , dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP 3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP 3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

    doi: 10.1016/j.jbc.2022.102264

    Figure Lengend Snippet: Phospholipid analogs differentially recovered TMEM16A. Soluble synthetic analogs of PIP 3 (diC8-PIP 3 ), PI(3,4)P 2 (diC8-PI(3,4)P 2 ), and PI(3,5)P 2 were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. A , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PIP 3 (N = 7), diC8-PI(3,4)P 2 (N = 6), or diC8-PI(3,5)P 2 (N = 5). Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PIP 3 ( B ), diC8-PI(3,4)P 2 ( C ), or diC8-PI(3,5)P 2 ( D ). ∗ represents p < 0.025 as determined by ANOVA and Tukey's HSD post hoc tests. diC8-PI(3,4)P 2 , dioctanoyl phosphatidylinositol 3,4-bisphosphate; diC8-PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; diC8-HSD, honestly significant difference; PIP 3, dioctanoyl phosphatidylinositol 3,4,5-trisphosphate; PI(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PI(3,5)P 2 , dioctanoyl phosphatidylinositol 3,5-bisphosphate; PIP 3, phosphatidyl 3,4,5-trisphosphate; TMEM16A, TransMEMbrane 16A.

    Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

    Techniques: Stable Transfection

    Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P ( A ), 100 μM diC8-PI5P ( B ), or 100 μM diC8-PI4P ( D ). C , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P 2 , as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

    doi: 10.1016/j.jbc.2022.102264

    Figure Lengend Snippet: Phospholipids with phosphates at position 4′ of the inositol ring recover current. Soluble synthetic analogs of PI3P, PI4P, and PI5P were applied to excised inside–out patches once current had stably rundown. Currents were recorded at −60 mV. Representative plots of normalized currents versus time, before and during application of 100 μM diC8-PI3P ( A ), 100 μM diC8-PI5P ( B ), or 100 μM diC8-PI4P ( D ). C , box plot distribution of the fold current recovered after the application of diC8-PI(4,5)P 2 (N = 9), diC8-PI3P (N = 7), diC8-PI4P (N = 7), or diC8-PI5P (N = 5). ∗ denotes p < 0.05 between indicated treatment and diC8-PI(4,5)P 2 , as determined by ANOVA and Tukey’s HSD post hoc tests. diC8-PI3P, dioctanoyl 3-monophosphate; diC8-PI4P, dioctanoyl 4-monophosphate; diC8-PI5P, dioctanoyl 5-monophosphate; HSD, honestly significant difference.

    Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

    Techniques: Stable Transfection

    Docking suggests key PI(4,5)P 2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P 2 or IP 3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A , position of diC8-PI(4,5)P 2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB ). B , detailed view of the hypothesized PI(4,5)P 2 –xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C , superposition of docked IP 3 (foreground) on the PI(4,5)P 2 –xTMEM16A (transparency) interaction. diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P 2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

    doi: 10.1016/j.jbc.2022.102264

    Figure Lengend Snippet: Docking suggests key PI(4,5)P 2 phosphate interactions with TMEM16A. Docking was performed with either diC8-PI(4,5)P 2 or IP 3 into a homology model of Xenopus laevis TMEM16A (xTMEM16A). A , position of diC8-PI(4,5)P 2 shown against the homology model of xTMEM16A. Lines indicating the position of the intracellular and extracellular boundaries of the plasma membrane were created using the OPM entry for mouse TMEM16A (PDB: 5OYB ). B , detailed view of the hypothesized PI(4,5)P 2 –xTMEM16A interaction. Interacting residues (E442, K446, R450, K592, and K912 from the other chain) and phosphates (positions 2′–5′) are highlighted. C , superposition of docked IP 3 (foreground) on the PI(4,5)P 2 –xTMEM16A (transparency) interaction. diC8-PI(4,5)P 2 , dioctanoyl phosphatidylinositol 4,5-bisphosphate; PDB, Protein Data Bank; PI(4,5)P 2, dioctanoyl phosphatidylinositol 4,5-bisphosphate; TMEM16A, TransMEMbrane 16A; xTMEM16A, Xenopus laevis TransMEMbrane 16A.

    Article Snippet: The following dioctanoyl phospholipids were obtained from Echelon Biosciences: diC8-PI3P, diC8-PI4P, diC8-PI5P, diC8-PI(3,4)P 2 , diC8-PI(4,5)P 2 , diC8-PI(3,5)P 2 , and diC8-PIP 3 .

    Techniques: