anti pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 3 5 p 2
    Anti Pi 3 5 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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    anti pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 3 5 p 2
    Anti Pi 3 5 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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    di c8 phosphoinositides  (Echelon Biosciences)


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    Echelon Biosciences di c8 phosphoinositides
    HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, <t>PtdIns(4,5)</t> P 2 .
    Di C8 Phosphoinositides, 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 "Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1"

    Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013396

    HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, PtdIns(4,5) P 2 .
    Figure Legend Snippet: HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, PtdIns(4,5) P 2 .

    Techniques Used: Isolation, In Vivo

    Thin layer chromatographic separation of the products of the in vitro phosphorylation reaction between PIP4Kάandendogenous phosphoinositides isolated from leaves (lane 2), stems (lane 3), flowers (lane 4) and siliques (lane 5). A PtdIns(4,5) P 2 standard is shown (lane 1) (panel a). HPLC analysis of deacylated products of the in vitro phosphorylation reaction between endogenous phosphoinositides (from leaves) and PIP4Kα. Deacylated PtdIns(3,4) P 2 and PtdIns(4,5) P 2 used as standards (panel b). The products of the in vitro phosphorylation reaction between endogenous phosphoinositides isolated from rosette leaves and PIP4Kα were incubated in the absence (lane 1) or presence (lane 2) of yeast YNK-5 phosphatase. The reaction products were analyzed by thin layer chromatography. The position of a PtdIns4 P standard is shown (lane 3) (panel c).
    Figure Legend Snippet: Thin layer chromatographic separation of the products of the in vitro phosphorylation reaction between PIP4Kάandendogenous phosphoinositides isolated from leaves (lane 2), stems (lane 3), flowers (lane 4) and siliques (lane 5). A PtdIns(4,5) P 2 standard is shown (lane 1) (panel a). HPLC analysis of deacylated products of the in vitro phosphorylation reaction between endogenous phosphoinositides (from leaves) and PIP4Kα. Deacylated PtdIns(3,4) P 2 and PtdIns(4,5) P 2 used as standards (panel b). The products of the in vitro phosphorylation reaction between endogenous phosphoinositides isolated from rosette leaves and PIP4Kα were incubated in the absence (lane 1) or presence (lane 2) of yeast YNK-5 phosphatase. The reaction products were analyzed by thin layer chromatography. The position of a PtdIns4 P standard is shown (lane 3) (panel c).

    Techniques Used: In Vitro, Isolation, Incubation, Thin Layer Chromatography

    Nuclear localisation of ePHD-GFP expressed alone. Arrows in all panels point to nuclei (panel a). ePHD-GFP co-expressed with wild-type AtMTM1-RFP causes depletion of the green nuclear signal (panel b). The consensus amino acids in the active AtMTM1 phosphatase site and the amino acid substitutions in mut1 and mut2 (panel c). Phosphoinositide 3′-phosphatase activity of recombinant wild type, mut1 and mut2 phosphatases. The Vmax values towards PtdIns(3,5) P 2 as a substrate are shown as the percentage of the wild-type (WT) ATMTM1 activity. Bars are s.d. from three measurements (panel d). Distribution of ePHD-GFP co-expressed with mut1AtMTM1-RFP show nuclear localizations of the green signal (panel e). Co-expression of ePHD-GFP with mut2AtMTM1-RFP. The green signal is largely distributed in the cytoplasm (panel f). Bars are 50 Pm.
    Figure Legend Snippet: Nuclear localisation of ePHD-GFP expressed alone. Arrows in all panels point to nuclei (panel a). ePHD-GFP co-expressed with wild-type AtMTM1-RFP causes depletion of the green nuclear signal (panel b). The consensus amino acids in the active AtMTM1 phosphatase site and the amino acid substitutions in mut1 and mut2 (panel c). Phosphoinositide 3′-phosphatase activity of recombinant wild type, mut1 and mut2 phosphatases. The Vmax values towards PtdIns(3,5) P 2 as a substrate are shown as the percentage of the wild-type (WT) ATMTM1 activity. Bars are s.d. from three measurements (panel d). Distribution of ePHD-GFP co-expressed with mut1AtMTM1-RFP show nuclear localizations of the green signal (panel e). Co-expression of ePHD-GFP with mut2AtMTM1-RFP. The green signal is largely distributed in the cytoplasm (panel f). Bars are 50 Pm.

    Techniques Used: Activity Assay, Recombinant, Expressing

    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:

    ptdins 3 5 p 2 dic16  (Echelon Biosciences)


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    Echelon Biosciences ptdins 3 5 p 2 dic16
    Ptdins 3 5 P 2 Dic16, 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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    ptdins 3 5 p 2 dic16  (Echelon Biosciences)


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    Echelon Biosciences ptdins 3 5 p 2 dic16
    Ptdins 3 5 P 2 Dic16, 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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    dic8 pi 3 5 p2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 5 p2
    Dic8 Pi 3 5 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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    pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 3 5 p 2
    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.
    Pi 3 5 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 "Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes"

    Article Title: Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31959-0

    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Concentration Assay, Two Tailed Test, Derivative Assay

    a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.

    Techniques Used: Concentration Assay, Fluorescence, Two Tailed Test

    pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences pi 3 5 p 2
    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.
    Pi 3 5 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 "Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes"

    Article Title: Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31959-0

    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Concentration Assay, Two Tailed Test, Derivative Assay

    a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.
    Figure Legend Snippet: a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.

    Techniques Used: Concentration Assay, Fluorescence, Two Tailed Test

    3 5 bisphosphate dic16  (Echelon Biosciences)


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    Echelon Biosciences 3 5 bisphosphate dic16
    3 5 Bisphosphate Dic16, 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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    Average 94 stars, based on 1 article reviews
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    3 5 bisphosphate dic16 - by Bioz Stars, 2023-01
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    dic8 pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 5 p 2
    (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates <t>diC8-PI3P,</t> <t>diC8-PI(3,5)P</t> 2 and <t>diC8-PI(4,5)P</t> 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.
    Dic8 Pi 3 5 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
    https://www.bioz.com/result/dic8 pi 3 5 p 2/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dic8 pi 3 5 p 2 - by Bioz Stars, 2023-01
    94/100 stars

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    1) Product Images from "Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane"

    Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

    Journal: bioRxiv

    doi: 10.1101/2022.07.26.501514

    (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates diC8-PI3P, diC8-PI(3,5)P 2 and diC8-PI(4,5)P 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.
    Figure Legend Snippet: (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates diC8-PI3P, diC8-PI(3,5)P 2 and diC8-PI(4,5)P 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.

    Techniques Used: Binding Assay, Staining, SDS Page, Negative Control

    (A) Gel filtration profile of Atg18-WT (black) and Atg18-FGGG (rose). Uncropped and unedited Coomassie-stained SDS-PAGE gels of pelletation assay used for preparing : (B) FGGG mutant (left) in comparison with Atg18-WT (left) and (C) Atg18 incubated with water (control), soluble DiC8-PI3P, DiC8-PI(3,5)P 2 and DiC8-PI(4,5)P 2 as indicated on top of the lanes.
    Figure Legend Snippet: (A) Gel filtration profile of Atg18-WT (black) and Atg18-FGGG (rose). Uncropped and unedited Coomassie-stained SDS-PAGE gels of pelletation assay used for preparing : (B) FGGG mutant (left) in comparison with Atg18-WT (left) and (C) Atg18 incubated with water (control), soluble DiC8-PI3P, DiC8-PI(3,5)P 2 and DiC8-PI(4,5)P 2 as indicated on top of the lanes.

    Techniques Used: Filtration, Staining, SDS Page, Mutagenesis, Incubation

    (A) Volume density rendering of a reconstructed tomogram containing Atg18-WT and PI(3,5)P 2 -containing large unilamellar vesicles (LUVs). (B) Central slice of the corresponding tomogram acquired with Volta phase plate (averaged five slices to display increased contrast). (C) Examples of rotational 2D classification after subvolume extraction of double-membrane subtomograms. The 60 Å thick membrane bilayers are spaced apart by 80 Å. (D) Initial subvolume average dominated by membrane density contributions. (E) Focused 3D classes after membrane subtraction reveal protein densities. (F) Refined structure of the selected 3D class (C1 symmetry) shows organized stacking of eight disc-shaped molecules. (G) Density fit with four Atg18-WT dimers formed by I-interface previously observed in Atg18 tubular assemblies and soluble dimers. The Atg18 molecule within the dimer is colored orange and magenta, respectively. The remaining six Atg18 molecules are colored grey. FRRG motif is highlighted in red and allows for direct binding of opposing membranes. (H) Slice through reconstructed tomogram reveals Atg18 dimer stacking with the approx. 45° angle relative to the membrane bilayer planes.
    Figure Legend Snippet: (A) Volume density rendering of a reconstructed tomogram containing Atg18-WT and PI(3,5)P 2 -containing large unilamellar vesicles (LUVs). (B) Central slice of the corresponding tomogram acquired with Volta phase plate (averaged five slices to display increased contrast). (C) Examples of rotational 2D classification after subvolume extraction of double-membrane subtomograms. The 60 Å thick membrane bilayers are spaced apart by 80 Å. (D) Initial subvolume average dominated by membrane density contributions. (E) Focused 3D classes after membrane subtraction reveal protein densities. (F) Refined structure of the selected 3D class (C1 symmetry) shows organized stacking of eight disc-shaped molecules. (G) Density fit with four Atg18-WT dimers formed by I-interface previously observed in Atg18 tubular assemblies and soluble dimers. The Atg18 molecule within the dimer is colored orange and magenta, respectively. The remaining six Atg18 molecules are colored grey. FRRG motif is highlighted in red and allows for direct binding of opposing membranes. (H) Slice through reconstructed tomogram reveals Atg18 dimer stacking with the approx. 45° angle relative to the membrane bilayer planes.

    Techniques Used: Binding Assay

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    Echelon Biosciences anti pi 3 5 p 2
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    Echelon Biosciences di c8 phosphoinositides
    HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, <t>PtdIns(4,5)</t> P 2 .
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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 ptdins 3 5 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 5 p2
    ( 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 pi 3 5 p 2
    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.
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    Echelon Biosciences 3 5 bisphosphate dic16
    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.
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    Echelon Biosciences dic8 pi 3 5 p 2
    (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates <t>diC8-PI3P,</t> <t>diC8-PI(3,5)P</t> 2 and <t>diC8-PI(4,5)P</t> 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.
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    Image Search Results


    HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, PtdIns(4,5) P 2 .

    Journal: PLoS ONE

    Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

    doi: 10.1371/journal.pone.0013396

    Figure Lengend Snippet: HPLC analysis of deacylated lipid products isolated from rosette leaves of in vivo radiolabeled whole plants with orthophosphate (300 µCi/ml) for 16 hours at room temperature. Peak identification: 1, phospholipids with positive charge; 2, phosphatidylinositol; 3, phosphatidic acid; 4, free inorganic phosphate; 5, PtdIns3 P ; 6, PtdIns4 P ; 7, PtdIns5 P ; 8, PtdIns(4,5) P 2 .

    Article Snippet: Di-C8 phosphoinositides (Echelon Biosciences Inc. catalogue numbers P-3008, P-5008, and P-3508) were used as substrates and PTEN lipid phosphatase (Echelon Biosciences Inc. catalogue number E-3000) was used as a positive control.

    Techniques: Isolation, In Vivo

    Thin layer chromatographic separation of the products of the in vitro phosphorylation reaction between PIP4Kάandendogenous phosphoinositides isolated from leaves (lane 2), stems (lane 3), flowers (lane 4) and siliques (lane 5). A PtdIns(4,5) P 2 standard is shown (lane 1) (panel a). HPLC analysis of deacylated products of the in vitro phosphorylation reaction between endogenous phosphoinositides (from leaves) and PIP4Kα. Deacylated PtdIns(3,4) P 2 and PtdIns(4,5) P 2 used as standards (panel b). The products of the in vitro phosphorylation reaction between endogenous phosphoinositides isolated from rosette leaves and PIP4Kα were incubated in the absence (lane 1) or presence (lane 2) of yeast YNK-5 phosphatase. The reaction products were analyzed by thin layer chromatography. The position of a PtdIns4 P standard is shown (lane 3) (panel c).

    Journal: PLoS ONE

    Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

    doi: 10.1371/journal.pone.0013396

    Figure Lengend Snippet: Thin layer chromatographic separation of the products of the in vitro phosphorylation reaction between PIP4Kάandendogenous phosphoinositides isolated from leaves (lane 2), stems (lane 3), flowers (lane 4) and siliques (lane 5). A PtdIns(4,5) P 2 standard is shown (lane 1) (panel a). HPLC analysis of deacylated products of the in vitro phosphorylation reaction between endogenous phosphoinositides (from leaves) and PIP4Kα. Deacylated PtdIns(3,4) P 2 and PtdIns(4,5) P 2 used as standards (panel b). The products of the in vitro phosphorylation reaction between endogenous phosphoinositides isolated from rosette leaves and PIP4Kα were incubated in the absence (lane 1) or presence (lane 2) of yeast YNK-5 phosphatase. The reaction products were analyzed by thin layer chromatography. The position of a PtdIns4 P standard is shown (lane 3) (panel c).

    Article Snippet: Di-C8 phosphoinositides (Echelon Biosciences Inc. catalogue numbers P-3008, P-5008, and P-3508) were used as substrates and PTEN lipid phosphatase (Echelon Biosciences Inc. catalogue number E-3000) was used as a positive control.

    Techniques: In Vitro, Isolation, Incubation, Thin Layer Chromatography

    Nuclear localisation of ePHD-GFP expressed alone. Arrows in all panels point to nuclei (panel a). ePHD-GFP co-expressed with wild-type AtMTM1-RFP causes depletion of the green nuclear signal (panel b). The consensus amino acids in the active AtMTM1 phosphatase site and the amino acid substitutions in mut1 and mut2 (panel c). Phosphoinositide 3′-phosphatase activity of recombinant wild type, mut1 and mut2 phosphatases. The Vmax values towards PtdIns(3,5) P 2 as a substrate are shown as the percentage of the wild-type (WT) ATMTM1 activity. Bars are s.d. from three measurements (panel d). Distribution of ePHD-GFP co-expressed with mut1AtMTM1-RFP show nuclear localizations of the green signal (panel e). Co-expression of ePHD-GFP with mut2AtMTM1-RFP. The green signal is largely distributed in the cytoplasm (panel f). Bars are 50 Pm.

    Journal: PLoS ONE

    Article Title: Phosphatidylinositol 5-Phosphate Links Dehydration Stress to the Activity of ARABIDOPSIS TRITHORAX-LIKE Factor ATX1

    doi: 10.1371/journal.pone.0013396

    Figure Lengend Snippet: Nuclear localisation of ePHD-GFP expressed alone. Arrows in all panels point to nuclei (panel a). ePHD-GFP co-expressed with wild-type AtMTM1-RFP causes depletion of the green nuclear signal (panel b). The consensus amino acids in the active AtMTM1 phosphatase site and the amino acid substitutions in mut1 and mut2 (panel c). Phosphoinositide 3′-phosphatase activity of recombinant wild type, mut1 and mut2 phosphatases. The Vmax values towards PtdIns(3,5) P 2 as a substrate are shown as the percentage of the wild-type (WT) ATMTM1 activity. Bars are s.d. from three measurements (panel d). Distribution of ePHD-GFP co-expressed with mut1AtMTM1-RFP show nuclear localizations of the green signal (panel e). Co-expression of ePHD-GFP with mut2AtMTM1-RFP. The green signal is largely distributed in the cytoplasm (panel f). Bars are 50 Pm.

    Article Snippet: Di-C8 phosphoinositides (Echelon Biosciences Inc. catalogue numbers P-3008, P-5008, and P-3508) were used as substrates and PTEN lipid phosphatase (Echelon Biosciences Inc. catalogue number E-3000) was used as a positive control.

    Techniques: Activity Assay, Recombinant, Expressing

    ( 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:

    a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes

    doi: 10.1038/s41467-022-31959-0

    Figure Lengend Snippet: a Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 30 µM) or a combination of the two (N+P) on Na + levels of individual SBFI-loaded HEK cells stably expressing TPC2 L11A/L12A . Each trace is the normalised fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. b Pooled data (mean ± sem) quantifying the rate of Na + influx from 4-8 experiments in response to the indicated concentration of agonists. n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). c , d Comparison of TPC2-A1-N and TPC2-A1-P responses on cytosolic Na + and Ca 2+ in TPC2 L11A/L12A -expressing cells loaded with SBFI and Fura-2, respectively. Data are mean ± sem from 4 to 10 experiments. e – h Effect of sequential agonist additions on currents from HEK cells stably expressing TPC2 L11A/L12A under bi-ionic conditions. Macropatches were stimulated with 10 µM TPC2-A1-N and 10 µM TPC2-A1-P ( e ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( g ) in the indicated order. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −100 mV and outward Na + currents at +100 mV from 3 to 6 experiments before (basal) and after agonist addition are shown in f and h . * p < 0.05, *** p = 0.0003, **** p < 0.0001, n.s. not significant (Paired t-test, two-tailed); * p = 0.04, ** p = 0.002, n.s. not significant (Unpaired t-test, two-tailed, in red). i – l Effect of agonist additions on currents from HEK cells stably expressing TPC2 in lysosomes under bi-ionic conditions. Cells were stimulated with 10 µM TPC2-A1-P or 10 µM TPC2-A1-N ( i ) or 1 µM PI(3,5)P 2 and 100 nM NAADP ( k ) either alone or in combination. Pooled data (mean ± sem) quantifying the inward Ca 2+ currents at −60 mV or outward Na + currents at +100 mV from 8 to 13 experiments in response to the agonists are shown in j and l , respectively. * p = 0.02, ** p = 0.005 (One-way ANOVA followed by Dunnett’s post hoc test) ( j ); * p = 0.02, **** p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test) ( l ); n.s. not significant (Unpaired t-test, two-tailed, j and l ). m , n Effect of TPC2 agonists on reversal potentials. Pooled data (mean ± sem from 3 to 13 experiments) quantifying the effect of TPC2 agonists on E rev in HEK cells stably expressing TPC2 L11A/L12A at the cell surface ( m ) or TPC2 in lysosomes ( n ). Values were derived from the bi-ionic experiments described in e – l . * p = 0.01, ** p = 0.007, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Tukey’s post hoc test) ( m ); *** p = 0.0005, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test) ( n ). Source data are provided as a Source Data file.

    Article Snippet: TPC2-A1-N, TPC2-A1-P, PI(3,5)P 2 (diC8 form; Echelon Biosciences) and NAADP (Bio-Techne) were applied by complete exchange of the cytoplasmic solution.

    Techniques: Stable Transfection, Expressing, Fluorescence, Concentration Assay, Two Tailed Test, Derivative Assay

    a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Segregated cation flux by TPC2 biases Ca 2+ signaling through lysosomes

    doi: 10.1038/s41467-022-31959-0

    Figure Lengend Snippet: a Effect of subthreshold concentration of TPC2-A1-N (10 µM), the indicated concentration of TPC2-A1-P or a combination on Ca 2+ levels of individual HeLa or SK-MEL-5 cells loaded with Fura-2. Data are presented as mean ± sem from 3 to 9 experiments. b , c Effect of subthreshold concentration of TPC2-A1-N (20 µM), TPC2-A1-P (20 µM) or a combination of the two on Ca 2+ levels of individual primary mouse pancreatic acinar cells loaded with Fura-2 b . Each trace is the normalized fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. Pooled data (mean ± sem) quantifying the peak change in ratio from 6 experiments are shown in c . **** p < 0.0001 (Unpaired t-test, two-tailed). d – f Effect of TPC2-A1-N (30 µM), TPC2-A1-P (30 µM) or a combination of the two on subcellular Ca 2+ levels of individual HEK cells loaded with Cal-520. Typical TIRF images with elementary events highlighted by circles are shown in d . Representative time courses of fluorescence changes from the centre of single tuff sites (1 × 1 μm) in response to the indicated agent. e Pooled data (mean ± sem) quantifying the number of events and sites detected per cell, and the peak response from 8 to 10 experiments are shown in f . * p < 0.05, **** p < 0.0001, n.s. not significant (One-way ANOVA followed by Dunnett’s post hoc test). g , h Effect of TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM) on lysosomal pH of individual SK-MEL-5 ( g ) or HeLa ( h ) cells loaded with fluorescein-dextran (FDx). Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view. The thicker trace is the average of the population. i, j , Effect of the indicated combinations of TPC2-A1-N and TPC2-A1-P on lysosomal pH of SK-MEL-5 cells i . Data are presented as mean ± sem from 3 to 5 experiments. Pooled data quantifying the peak change in ratio from multiple experiments using the indicated cell type and agonist combination are shown in j . * p = 0.02, ** p < 0.01 (One-way ANOVA followed by Dunnett’s post hoc test). k – m Effect of TPC2-A1-N and TPC2-A1-P on lysosomal motility. Images show maximum projections of motility calculated from differences in pixel-wise intensity on a frame-by-frame basis over an early (120–240 s) and late (570−690 s) period following addition of TPC2-A1-N (30 µM) or TPC2-A1-P (60 µM) to HeLa cells k . Intense signals represent large changes over time equating to more lysosome movement. Full time-courses presented as mean ± sem from 3 to 8 experiments in response to the indicated agonist combination are shown in l . Pooled data quantifying motility at 700 s from multiple experiments using the indicated cell type and agonist combination m . * p < 0.05, ** p = 0.008 (One-way ANOVA followed by Dunnett’s post hoc test). n Model showing that NAADP and PI(3,5)P 2 work in a synergistic manner to selectively optimize Ca 2+ signalling from lysosomes to regulate lysosomal pH and motility leaving Na + signals unperturbed. Source data are provided as a Source Data file.

    Article Snippet: TPC2-A1-N, TPC2-A1-P, PI(3,5)P 2 (diC8 form; Echelon Biosciences) and NAADP (Bio-Techne) were applied by complete exchange of the cytoplasmic solution.

    Techniques: Concentration Assay, Fluorescence, Two Tailed Test

    (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates diC8-PI3P, diC8-PI(3,5)P 2 and diC8-PI(4,5)P 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.

    Journal: bioRxiv

    Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

    doi: 10.1101/2022.07.26.501514

    Figure Lengend Snippet: (A) Atg18-PR72AA refined atomic model with surface colored by electrostatics, FRRG motif in pink and phosphate binding sites from aligned PDB-ID 5LTD. (B) Diamond shaped tetramer unit (green) with FRRG motifs (red) and their adjacent phosphate binding sites, which are buried in Atg18 tubes. (C) Pelletation assay (top) and negative stain of pellet fractions from Atg18-WT (left) and Atg18-FGGG (right) (D) Top. Chemical formulas of soluble PIP derivates diC8-PI3P, diC8-PI(3,5)P 2 and diC8-PI(4,5)P 2 used in binding experiments. Bottom. Negative stain electron micrographs of Atg18 nontreated or treated with 4x molar excess of the indicated PIP derivate preventing tube formation. (E) Pelletation assay with corresponding SDS-PAGE of the supernatant (SN) and the pellet (Pe) fraction and the negative stain EM micrographs of two pellets. (F) Tubes from the negative control were treated with PIP derivates. Only PI(3,5)P 2 resulted in tube disassembly.

    Article Snippet: To test the effect of soluble lipids on Atg18 tube formation, diC8-PI3P and diC8-PI(3,5)P 2 (Echelon) were reconstituted in water (1 mM final concentration) and added in 4x molar excess to Atg18 in gel filtration buffer prior to the dialysis into tube buffer.

    Techniques: Binding Assay, Staining, SDS Page, Negative Control

    (A) Gel filtration profile of Atg18-WT (black) and Atg18-FGGG (rose). Uncropped and unedited Coomassie-stained SDS-PAGE gels of pelletation assay used for preparing : (B) FGGG mutant (left) in comparison with Atg18-WT (left) and (C) Atg18 incubated with water (control), soluble DiC8-PI3P, DiC8-PI(3,5)P 2 and DiC8-PI(4,5)P 2 as indicated on top of the lanes.

    Journal: bioRxiv

    Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

    doi: 10.1101/2022.07.26.501514

    Figure Lengend Snippet: (A) Gel filtration profile of Atg18-WT (black) and Atg18-FGGG (rose). Uncropped and unedited Coomassie-stained SDS-PAGE gels of pelletation assay used for preparing : (B) FGGG mutant (left) in comparison with Atg18-WT (left) and (C) Atg18 incubated with water (control), soluble DiC8-PI3P, DiC8-PI(3,5)P 2 and DiC8-PI(4,5)P 2 as indicated on top of the lanes.

    Article Snippet: To test the effect of soluble lipids on Atg18 tube formation, diC8-PI3P and diC8-PI(3,5)P 2 (Echelon) were reconstituted in water (1 mM final concentration) and added in 4x molar excess to Atg18 in gel filtration buffer prior to the dialysis into tube buffer.

    Techniques: Filtration, Staining, SDS Page, Mutagenesis, Incubation

    (A) Volume density rendering of a reconstructed tomogram containing Atg18-WT and PI(3,5)P 2 -containing large unilamellar vesicles (LUVs). (B) Central slice of the corresponding tomogram acquired with Volta phase plate (averaged five slices to display increased contrast). (C) Examples of rotational 2D classification after subvolume extraction of double-membrane subtomograms. The 60 Å thick membrane bilayers are spaced apart by 80 Å. (D) Initial subvolume average dominated by membrane density contributions. (E) Focused 3D classes after membrane subtraction reveal protein densities. (F) Refined structure of the selected 3D class (C1 symmetry) shows organized stacking of eight disc-shaped molecules. (G) Density fit with four Atg18-WT dimers formed by I-interface previously observed in Atg18 tubular assemblies and soluble dimers. The Atg18 molecule within the dimer is colored orange and magenta, respectively. The remaining six Atg18 molecules are colored grey. FRRG motif is highlighted in red and allows for direct binding of opposing membranes. (H) Slice through reconstructed tomogram reveals Atg18 dimer stacking with the approx. 45° angle relative to the membrane bilayer planes.

    Journal: bioRxiv

    Article Title: Structural plasticity of Atg18 oligomers: organization of assembled tubes and scaffolds at the isolation membrane

    doi: 10.1101/2022.07.26.501514

    Figure Lengend Snippet: (A) Volume density rendering of a reconstructed tomogram containing Atg18-WT and PI(3,5)P 2 -containing large unilamellar vesicles (LUVs). (B) Central slice of the corresponding tomogram acquired with Volta phase plate (averaged five slices to display increased contrast). (C) Examples of rotational 2D classification after subvolume extraction of double-membrane subtomograms. The 60 Å thick membrane bilayers are spaced apart by 80 Å. (D) Initial subvolume average dominated by membrane density contributions. (E) Focused 3D classes after membrane subtraction reveal protein densities. (F) Refined structure of the selected 3D class (C1 symmetry) shows organized stacking of eight disc-shaped molecules. (G) Density fit with four Atg18-WT dimers formed by I-interface previously observed in Atg18 tubular assemblies and soluble dimers. The Atg18 molecule within the dimer is colored orange and magenta, respectively. The remaining six Atg18 molecules are colored grey. FRRG motif is highlighted in red and allows for direct binding of opposing membranes. (H) Slice through reconstructed tomogram reveals Atg18 dimer stacking with the approx. 45° angle relative to the membrane bilayer planes.

    Article Snippet: To test the effect of soluble lipids on Atg18 tube formation, diC8-PI3P and diC8-PI(3,5)P 2 (Echelon) were reconstituted in water (1 mM final concentration) and added in 4x molar excess to Atg18 in gel filtration buffer prior to the dialysis into tube buffer.

    Techniques: Binding Assay