pi 3 5 p 2 dic8 p4508  (Echelon Biosciences)


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    Echelon Biosciences pi 3 5 p 2 dic8 p4508
    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.
    Pi 3 5 P 2 Dic8 P4508, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling"

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033889

    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.
    Figure Legend Snippet: ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

    Techniques Used: Injection, Expressing, Dominant Negative Mutation

    Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.
    Figure Legend Snippet: Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

    Techniques Used:

    pi 3 5 p 2 dic8 p4508  (Echelon Biosciences)


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    Echelon Biosciences pi 3 5 p 2 dic8 p4508
    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.
    Pi 3 5 P 2 Dic8 P4508, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling"

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033889

    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.
    Figure Legend Snippet: ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

    Techniques Used: Injection, Expressing, Dominant Negative Mutation

    Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.
    Figure Legend Snippet: Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

    Techniques Used:

    eitherptdins 3 4 p 2 dic16  (Echelon Biosciences)


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    Echelon Biosciences eitherptdins 3 4 p 2 dic16
    Eitherptdins 3 4 P 2 Dic16, 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 pi 3 4 p2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 4 p2
    MptpB and related phosphatases have both protein phosphatase and lipid phosphatase activity . ( A ) Specific activity (SA) for His-MptpB WT, Lmo1800 WT, Lmo1935 WT, His-LM1 and His-TbPTP1 towards phosphorylated peptide substrates tested. ( B ) Specific activity of the same proteins towards PI substrates is shown. None of the phosphatases had activity on <t>PI(3,4)P2,</t> PI(4,5)P2 or PI(3,4,5)P3. Error bars indicate SEM.
    Dic8 Pi 3 4 P2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis"

    Article Title: A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-457

    MptpB and related phosphatases have both protein phosphatase and lipid phosphatase activity . ( A ) Specific activity (SA) for His-MptpB WT, Lmo1800 WT, Lmo1935 WT, His-LM1 and His-TbPTP1 towards phosphorylated peptide substrates tested. ( B ) Specific activity of the same proteins towards PI substrates is shown. None of the phosphatases had activity on PI(3,4)P2, PI(4,5)P2 or PI(3,4,5)P3. Error bars indicate SEM.
    Figure Legend Snippet: MptpB and related phosphatases have both protein phosphatase and lipid phosphatase activity . ( A ) Specific activity (SA) for His-MptpB WT, Lmo1800 WT, Lmo1935 WT, His-LM1 and His-TbPTP1 towards phosphorylated peptide substrates tested. ( B ) Specific activity of the same proteins towards PI substrates is shown. None of the phosphatases had activity on PI(3,4)P2, PI(4,5)P2 or PI(3,4,5)P3. Error bars indicate SEM.

    Techniques Used: Activity Assay

    A. P-loop composition of different lipid phosphatase activity . Two groups of lipid phosphatases can be assigned based on the different amino acid composition of the active site P-loop. Group 1 contains 3 or more basic residues and a conserved K in position two, while Group 2 have 1 or 2 basic residues and none in position 2 . B. Effect of mutagenesis of P-loop residues on the catalytic profile . Specific activity (SA) for WT and mutants of MptpB, Lm1800 and LM1 enzyme using different phosphoinositides as substrates. Addition of a second basic residue in the P-loop increases activity towards di-phosphorylated PIs and results in new activity for MptpB towards PI(3,4,5)P3. Error bars indicate SEM.
    Figure Legend Snippet: A. P-loop composition of different lipid phosphatase activity . Two groups of lipid phosphatases can be assigned based on the different amino acid composition of the active site P-loop. Group 1 contains 3 or more basic residues and a conserved K in position two, while Group 2 have 1 or 2 basic residues and none in position 2 . B. Effect of mutagenesis of P-loop residues on the catalytic profile . Specific activity (SA) for WT and mutants of MptpB, Lm1800 and LM1 enzyme using different phosphoinositides as substrates. Addition of a second basic residue in the P-loop increases activity towards di-phosphorylated PIs and results in new activity for MptpB towards PI(3,4,5)P3. Error bars indicate SEM.

    Techniques Used: Activity Assay, Mutagenesis

    dic8 pi 3 4 p 2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 4 p 2
    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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    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:

    phosphatidylinositol 3 4 bisphosphate pi 3 4 p2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences phosphatidylinositol 3 4 bisphosphate pi 3 4 p2
    Phosphatidylinositol 3 4 Bisphosphate Pi 3 4 P2, 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 3 4 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 3 4 p 2
    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.
    Pi 3 4 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia"

    Article Title: Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia

    Journal: iScience

    doi: 10.1016/j.isci.2022.104170

    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.
    Figure Legend Snippet: K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.

    Techniques Used: Activity Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Generated, FP Assay, Concentration Assay

    16:0/18  (Echelon Biosciences)


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    pi 4 5 p2  (Echelon Biosciences)


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    Echelon Biosciences pi 3 4 p 2
    Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .
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    1) Product Images from "Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases"

    Article Title: Class I PI3K Provide Lipid Substrate in T Cell Autophagy Through Linked Activity of Inositol Phosphatases

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.709398

    Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .
    Figure Legend Snippet: Exogenous PI(3,4)P 2 alters the autophagic profile and is converted to PI(3)P. ( A , left panel) Histograms of relative levels of PI(3)P and PI(3,4)P 2 CD4 T cells were loaded with 10 μM PI(3,4)P 2 and stimulated with 1 μg/mL soluble anti-CD3 and anti-CD28 for 24 and 120 h. PI(3,4)P 2 is largely converted to PI(3)P only after TCR stimulation. ( A , right panel) AVO formation of CD4 T cells loaded with PI(3,4)P 2 . Exogenous PI(3,4)P 2 effects an accelerated autophagic profile that is resolved by 120 h compared to unloaded controls. (B) Quantification of phosphatidyl inositol levels from CD4 T cells left untreated or loaded with PI(3,4)P 2 and stimulated for 24 h as in (A) . Data represent 4 replicates from 3 independent experiments. (C) Fluorescence microscopy of phosphatidylinositol species in T cells activated with plate-bound anti-CD3 and anti-CD28 for the indicated time periods. (D) Quantitation of colocalization of phosphatidylinositol species from (C) .

    Techniques Used: Fluorescence, Microscopy, Quantitation Assay

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    Echelon Biosciences pi 3 5 p 2 dic8 p4508
    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.
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    Echelon Biosciences eitherptdins 3 4 p 2 dic16
    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.
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    Echelon Biosciences dic8 pi 3 4 p2
    MptpB and related phosphatases have both protein phosphatase and lipid phosphatase activity . ( A ) Specific activity (SA) for His-MptpB WT, Lmo1800 WT, Lmo1935 WT, His-LM1 and His-TbPTP1 towards phosphorylated peptide substrates tested. ( B ) Specific activity of the same proteins towards PI substrates is shown. None of the phosphatases had activity on <t>PI(3,4)P2,</t> PI(4,5)P2 or PI(3,4,5)P3. Error bars indicate SEM.
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    Echelon Biosciences dic8 pi 3 4 p 2
    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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    Echelon Biosciences phosphatidylinositol 3 4 bisphosphate pi 3 4 p2
    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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    Echelon Biosciences pi 3 4 p 2
    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.
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    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.
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    Echelon Biosciences pi 4 5 p2
    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.
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    Echelon Biosciences ptdins 3 4 p2 dic16 p 3416
    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.
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    ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

    Journal: PLoS ONE

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

    doi: 10.1371/journal.pone.0033889

    Figure Lengend Snippet: ( A ) GluA1 current amplitudes in oocytes before and after acute injection of a water-soluble analog of PI(3,5)P 2 . Significant change (p<0.05) is indicated by * (p = 0.0015). ( B ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with Rab11 or the dominant negative form of Rab11 (Rab11DN), before and after acute injection of a water-soluble analog of PI (3,5)P 2 . Numbers of oocytes are n = 7–28. Significant change (p<0.05) is indicated by * (p = 0.025). ( C ) GluA1 current amplitudes in oocytes expressing GluA1 or combinations of GluA1 with SGK3, myosin Vb, or the mutated form of myosin Vb (myosin del). Number of oocytes are n = 14–28.

    Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

    Techniques: Injection, Expressing, Dominant Negative Mutation

    Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

    Journal: PLoS ONE

    Article Title: Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

    doi: 10.1371/journal.pone.0033889

    Figure Lengend Snippet: Upon stimulation of NMDA receptors SGK3 is transcriptionally upregulated. SGK3 in turn phosphorylates/activates PIKfyve, which leads to local production of PI(3,5)P 2 in PIKfyve-containing recycling vesicles. PI(3,5)P 2 in turn stimulates Rab11-dependent plasma membrane-directed trafficking of GluA1-containing vesicles.

    Article Snippet: PI(3,5)P 2 -DIC8 (P4508) was purchased from Echelon Bioscience Inc. (Salt Lake City, USA).

    Techniques:

    MptpB and related phosphatases have both protein phosphatase and lipid phosphatase activity . ( A ) Specific activity (SA) for His-MptpB WT, Lmo1800 WT, Lmo1935 WT, His-LM1 and His-TbPTP1 towards phosphorylated peptide substrates tested. ( B ) Specific activity of the same proteins towards PI substrates is shown. None of the phosphatases had activity on PI(3,4)P2, PI(4,5)P2 or PI(3,4,5)P3. Error bars indicate SEM.

    Journal: BMC Genomics

    Article Title: A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

    doi: 10.1186/1471-2164-11-457

    Figure Lengend Snippet: MptpB and related phosphatases have both protein phosphatase and lipid phosphatase activity . ( A ) Specific activity (SA) for His-MptpB WT, Lmo1800 WT, Lmo1935 WT, His-LM1 and His-TbPTP1 towards phosphorylated peptide substrates tested. ( B ) Specific activity of the same proteins towards PI substrates is shown. None of the phosphatases had activity on PI(3,4)P2, PI(4,5)P2 or PI(3,4,5)P3. Error bars indicate SEM.

    Article Snippet: The malachite green assay [ ] was used to determine the amount of free phosphate during the dephosphorylation assays with a range of substrates: phospho-Tyr peptides from EGFR (DADEpYLIPQQG) and insulin receptor (TRDIpYETDYYRK), phospho-Ser peptide (RRApSVA), phospho-Thr peptide (KRpTIRR) (Alta Bioscience, University of Birmingham, http://www.altabioscience.bham.ac.uk ), pNPP (Sigma), and the PIs diC8-PI(3)P, diC8-PI(3,4)P2, diC8-PI(3,5)P2, diC8-PI(4)P, diC8-PI(4,5)P2, diC8-PI(5)P and diC8-PI(3,4,5)P3 (Echelon Bioscience), phospho-amino acids, adenosine 5' monophosphate, inosine 5'-monophosphophate, phosphorylcholine chloride, phosphorylethanolamine, glycerol 2-phosphate and sodium pyrophosphate (Sigma).

    Techniques: Activity Assay

    A. P-loop composition of different lipid phosphatase activity . Two groups of lipid phosphatases can be assigned based on the different amino acid composition of the active site P-loop. Group 1 contains 3 or more basic residues and a conserved K in position two, while Group 2 have 1 or 2 basic residues and none in position 2 . B. Effect of mutagenesis of P-loop residues on the catalytic profile . Specific activity (SA) for WT and mutants of MptpB, Lm1800 and LM1 enzyme using different phosphoinositides as substrates. Addition of a second basic residue in the P-loop increases activity towards di-phosphorylated PIs and results in new activity for MptpB towards PI(3,4,5)P3. Error bars indicate SEM.

    Journal: BMC Genomics

    Article Title: A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

    doi: 10.1186/1471-2164-11-457

    Figure Lengend Snippet: A. P-loop composition of different lipid phosphatase activity . Two groups of lipid phosphatases can be assigned based on the different amino acid composition of the active site P-loop. Group 1 contains 3 or more basic residues and a conserved K in position two, while Group 2 have 1 or 2 basic residues and none in position 2 . B. Effect of mutagenesis of P-loop residues on the catalytic profile . Specific activity (SA) for WT and mutants of MptpB, Lm1800 and LM1 enzyme using different phosphoinositides as substrates. Addition of a second basic residue in the P-loop increases activity towards di-phosphorylated PIs and results in new activity for MptpB towards PI(3,4,5)P3. Error bars indicate SEM.

    Article Snippet: The malachite green assay [ ] was used to determine the amount of free phosphate during the dephosphorylation assays with a range of substrates: phospho-Tyr peptides from EGFR (DADEpYLIPQQG) and insulin receptor (TRDIpYETDYYRK), phospho-Ser peptide (RRApSVA), phospho-Thr peptide (KRpTIRR) (Alta Bioscience, University of Birmingham, http://www.altabioscience.bham.ac.uk ), pNPP (Sigma), and the PIs diC8-PI(3)P, diC8-PI(3,4)P2, diC8-PI(3,5)P2, diC8-PI(4)P, diC8-PI(4,5)P2, diC8-PI(5)P and diC8-PI(3,4,5)P3 (Echelon Bioscience), phospho-amino acids, adenosine 5' monophosphate, inosine 5'-monophosphophate, phosphorylcholine chloride, phosphorylethanolamine, glycerol 2-phosphate and sodium pyrophosphate (Sigma).

    Techniques: Activity Assay, Mutagenesis

    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:

    K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.

    Journal: iScience

    Article Title: Discovery of a novel SHIP1 agonist that promotes degradation of lipid-laden phagocytic cargo by microglia

    doi: 10.1016/j.isci.2022.104170

    Figure Lengend Snippet: K306 has biological activity consistent with SHIP1 agonistic activity and is a more potent agonist than the pelorol AQX-MN100 (A) IL-6 and (B)TNF-α production by BV2 microglia cells stimulated with LPS for 6 h or 2 h, respectively, as measured from supernatants by ELISA. (Representative results of two independent comparisons of all potential agonists at 5μM). All cells were treated with agonists or vehicle control 1 h before LPS challenge. Bars indicate mean ±SEM. Statistical analysis was performed with one-way ANOVA with Dunnett correction for multiple comparisons versus control (DMSO), ∗∗p < 0.01, ∗p < 0.05). 5′ fluorescence polarization Assay (FP) to measure PtdIns(3,4,5)P 3 5′ phosphatase activity on (C) tSHIP1 with K306 and MN-100 and (D) SHIP1-Enzyme (S1-Enz) and SHIP2-Enzyme (S2-Enz) with K306. EC 50 of K306 and MN-100 on tSHIP1 and the EC 50 of K306 on S1-Enz and S2-Enz were calculated by incubating dilutions of K306 or MN-100 with either enzyme for 20 min at 37°C. PI(3,4)P 2 generated by the SHIP enzymes is then measured using the FP assay (Echelon Biosciences). Control reactions include probe alone (PA), where all the probe is free rotating and thus not polarized and no-enzyme (NE) control, where the probe is not displaced from the detector by enzymatically generated PI(3,4)P 2 and highly polarized. Shown is one of 2 independent experiments with 6 replicate wells/compound concentration. EC 50 was calculated with Prizm version 9.3.1, using Nonlinear regression [Agonist] vs. response (three parameters), using Robust Fitting method, Medium Convergence Criteria, No Weighting and Considering every Y replicate as an individual point.

    Article Snippet: FP Assay for detection of PI(3,4)P 2 (Echelon Biosciences) was performed using tSHIP1 , SHIP1-Enz or SHIP2-Enz with serial dilutions of K306 (in DMSO) or AQX-MN100 (in ETOH) according to manufacturer’s recommendation as previously described ( ).

    Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Generated, FP Assay, Concentration Assay