phosphatidylinositol 4 5 bisphosphate  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 4 5 bisphosphate
    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).
    Phosphatidylinositol 4 5 Bisphosphate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway"

    Article Title: PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway

    Journal: bioRxiv

    doi: 10.1101/2024.02.23.581751

    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).
    Figure Legend Snippet: PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).

    Techniques Used: Incubation, MANN-WHITNEY

    d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl  (Echelon Biosciences)


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    Echelon Biosciences d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl
    D Myo Phosphatidylinositol 4 5 Bisphosphate Ptdins 4 5 P2 D Sn 1 2 Di O Octanoylglyceryl, 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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    d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl  (Echelon Biosciences)


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    Echelon Biosciences d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl
    D Myo Phosphatidylinositol 4 5 Bisphosphate Ptdins 4 5 P2 D Sn 1 2 Di O Octanoylglyceryl, 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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    phosphoinositol 4 5 bisphosphate pip2  (Echelon Biosciences)


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    Echelon Biosciences phosphoinositol 4 5 bisphosphate pip2
    Phosphoinositol 4 5 Bisphosphate Pip2, 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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    4 5 bisphosphate  (Echelon Biosciences)


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    Echelon Biosciences 4 5 bisphosphate
    4 5 Bisphosphate, 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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    d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl  (Echelon Biosciences)


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    Echelon Biosciences d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl
    D Myo Phosphatidylinositol 4 5 Bisphosphate Ptdins 4 5 P2 D Sn 1 2 Di O Octanoylglyceryl, 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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    d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl  (Echelon Biosciences)


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    Echelon Biosciences d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl
    D Myo Phosphatidylinositol 4 5 Bisphosphate Ptdins 4 5 P2 D Sn 1 2 Di O Octanoylglyceryl, 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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    l α phosphatidylinositol 4 5 bisphosphate  (Echelon Biosciences)


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    Echelon Biosciences l α phosphatidylinositol 4 5 bisphosphate
    (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).
    L α Phosphatidylinositol 4 5 Bisphosphate, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback"

    Article Title: Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback

    Journal: bioRxiv

    doi: 10.1101/2023.09.04.556152

    (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).
    Figure Legend Snippet: (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).

    Techniques Used: Mutagenesis, Activity Assay, Concentration Assay, Membrane, Binding Assay

    (A) Cartoon showing the structure of the mNG-PIP5K specificity loop-PLCδ chimeric protein. The expected PIP lipid binding interactions are indicated. (B) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing either 4% PI(4)P or 4% PI(5)P. (C) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing 4% PI(4,5)P 2 . (D) Sequence alignment of PIP5K and PIP4K specificity loops. (E) Cartoon showing the PIP4K to PIP5K specificity loop swap. (F) Quantification of the fold increase in localization of Cy5-PIP4K and Cy5-PIP4K(5K-SL) on membranes containing different molar concentrations of PI(4,5)P 2 . The fold change was calculated relative to the measured membrane density in the absence of PI(4,5)P 2 . (G) Single molecule dwell time distributions of Cy5-PIP4K and Cy5-PIP4K(5K-SL) measured on membranes containing 4% PI(4,5)P 2 . (H) Plots showing the cumulative membrane binding events measured in the presence of 20-80 pM Cy5-PIP4K. The slope of each curve was calculated by linear regression yielding a binding frequency for each concentration of Cy5-PIP4K. (I) Experimental measure of membrane binding frequency ( k ON ) in the presence of 20-80 pM Cy5-PIP4K or 5-10 pM Cy5-PIP4K(5K-SL).
    Figure Legend Snippet: (A) Cartoon showing the structure of the mNG-PIP5K specificity loop-PLCδ chimeric protein. The expected PIP lipid binding interactions are indicated. (B) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing either 4% PI(4)P or 4% PI(5)P. (C) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing 4% PI(4,5)P 2 . (D) Sequence alignment of PIP5K and PIP4K specificity loops. (E) Cartoon showing the PIP4K to PIP5K specificity loop swap. (F) Quantification of the fold increase in localization of Cy5-PIP4K and Cy5-PIP4K(5K-SL) on membranes containing different molar concentrations of PI(4,5)P 2 . The fold change was calculated relative to the measured membrane density in the absence of PI(4,5)P 2 . (G) Single molecule dwell time distributions of Cy5-PIP4K and Cy5-PIP4K(5K-SL) measured on membranes containing 4% PI(4,5)P 2 . (H) Plots showing the cumulative membrane binding events measured in the presence of 20-80 pM Cy5-PIP4K. The slope of each curve was calculated by linear regression yielding a binding frequency for each concentration of Cy5-PIP4K. (I) Experimental measure of membrane binding frequency ( k ON ) in the presence of 20-80 pM Cy5-PIP4K or 5-10 pM Cy5-PIP4K(5K-SL).

    Techniques Used: Binding Assay, Sequencing, Membrane, Concentration Assay

    (A) AlphaFold structure of human PIP5KB validated against zPIP5Kα (5E3U.pdb). Structure highlights the specificity loop (red) and the PIP lipid (or substrate) Binding Motif (PIPBM, blue). (B) Single molecule dwell time distributions of AF647-PIP5KB measured in the buffer containing either 1 mM ATP or ADP. (C) Bulk membrane recruitment of either 25nM mNG-PIP5KB or mNG-PIP5KB (K205A/ R211A). (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Single molecule dwell time distribution comparing mNG-PIP5KB and mNG-PIP5KB (K205A/R211A). Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (E) Representative TIRF-M images showing the localization of the mEos3.2-PIP5KB (WT and K205A/R211A) expressed in HEK293T cells. (F) Kymograph showing the single molecule plasma membrane binding dynamics of mEos3.2-PIP5KB (WT and K205A/R211A) in HEK293T cells. (G) Single molecule dwell time distributions for mEos3.2-PIP5KB (WT and K205A/R211A) measured in HEK293T cells. (H) Schematic showing SpyTag-PIP5KB(K205A/ R211A) and SpyCatcher kinase activity assay. (I) Kinetics of lipid phosphorylation measured for membrane tethered and non-tethered SpyTag-PIP5KB(K205A/R211A). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ.
    Figure Legend Snippet: (A) AlphaFold structure of human PIP5KB validated against zPIP5Kα (5E3U.pdb). Structure highlights the specificity loop (red) and the PIP lipid (or substrate) Binding Motif (PIPBM, blue). (B) Single molecule dwell time distributions of AF647-PIP5KB measured in the buffer containing either 1 mM ATP or ADP. (C) Bulk membrane recruitment of either 25nM mNG-PIP5KB or mNG-PIP5KB (K205A/ R211A). (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Single molecule dwell time distribution comparing mNG-PIP5KB and mNG-PIP5KB (K205A/R211A). Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (E) Representative TIRF-M images showing the localization of the mEos3.2-PIP5KB (WT and K205A/R211A) expressed in HEK293T cells. (F) Kymograph showing the single molecule plasma membrane binding dynamics of mEos3.2-PIP5KB (WT and K205A/R211A) in HEK293T cells. (G) Single molecule dwell time distributions for mEos3.2-PIP5KB (WT and K205A/R211A) measured in HEK293T cells. (H) Schematic showing SpyTag-PIP5KB(K205A/ R211A) and SpyCatcher kinase activity assay. (I) Kinetics of lipid phosphorylation measured for membrane tethered and non-tethered SpyTag-PIP5KB(K205A/R211A). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ.

    Techniques Used: Binding Assay, Membrane, Kinase Assay

    (A) Single molecule dwell time distributions measured in the presence of AF647-PIP5KB on SLBs containing 2% of the indicated PIP lipid. (B) Kinetics of PIP5KB dependent lipid phosphorylation measured in the presence of 0-20% DOPS. All membranes contain 2% PI(4)P and reactions were monitored using 20 nM Cy3-PLCδ. Kinetics were quantified by comparing the half-time for reaction completion. (C) Quantification of the fold increase of membrane recruitment measured in the presence of 15 nM mNG-PIP5KB on SLBs containing 0-20% DOPS. The fold increase in membrane binding we calculated relative to the membrane intensity of mNG-PIP5K measured in the absence of DOPS. (D) Single molecule dwell time analysis of AF647-PIP5KB in the presence of 20% DOPS or 2% PI(4,5)P 2 . (E) Representative TIRF-M images showing the membrane localization of 15 nM mNG-PIP5K or mNG-PIP5K (K205A/R211A). Membrane composition: 20% DOPS, 80% DOPC. (F) Quantification of bulk recruitment data shown in (E).
    Figure Legend Snippet: (A) Single molecule dwell time distributions measured in the presence of AF647-PIP5KB on SLBs containing 2% of the indicated PIP lipid. (B) Kinetics of PIP5KB dependent lipid phosphorylation measured in the presence of 0-20% DOPS. All membranes contain 2% PI(4)P and reactions were monitored using 20 nM Cy3-PLCδ. Kinetics were quantified by comparing the half-time for reaction completion. (C) Quantification of the fold increase of membrane recruitment measured in the presence of 15 nM mNG-PIP5KB on SLBs containing 0-20% DOPS. The fold increase in membrane binding we calculated relative to the membrane intensity of mNG-PIP5K measured in the absence of DOPS. (D) Single molecule dwell time analysis of AF647-PIP5KB in the presence of 20% DOPS or 2% PI(4,5)P 2 . (E) Representative TIRF-M images showing the membrane localization of 15 nM mNG-PIP5K or mNG-PIP5K (K205A/R211A). Membrane composition: 20% DOPS, 80% DOPC. (F) Quantification of bulk recruitment data shown in (E).

    Techniques Used: Membrane, Binding Assay

    (A) Structure of PIP5K (5E3U.pdb) showing the position of residues that are important for catalysis. (B) Representative TIRF-M images showing the localization of mNG-PIP5KB (WT and mutants). (C) Single molecule dwell time distributions for mNG-PIP5KB (WT and mutants) measured. (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Bulk membrane recruitment measured by TIRF-M in the presence of 20 nM mNG-PIP5KB (WT and mutants). Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (E-F) Lipid kinase activity measured using TIRF-M in the presence of 10 nM PIP5KB (WT and mutants). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ. (E) Membrane composition: 96% DOPC, 4% PI(4)P. (F) Membrane composition: 78% DOPC, 2% PI(4)P, 20% DOPS.
    Figure Legend Snippet: (A) Structure of PIP5K (5E3U.pdb) showing the position of residues that are important for catalysis. (B) Representative TIRF-M images showing the localization of mNG-PIP5KB (WT and mutants). (C) Single molecule dwell time distributions for mNG-PIP5KB (WT and mutants) measured. (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Bulk membrane recruitment measured by TIRF-M in the presence of 20 nM mNG-PIP5KB (WT and mutants). Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (E-F) Lipid kinase activity measured using TIRF-M in the presence of 10 nM PIP5KB (WT and mutants). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ. (E) Membrane composition: 96% DOPC, 4% PI(4)P. (F) Membrane composition: 78% DOPC, 2% PI(4)P, 20% DOPS.

    Techniques Used: Membrane, Activity Assay

    4 5 bisphosphate dic8  (Echelon Biosciences)


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    Echelon Biosciences 4 5 bisphosphate dic8
    4 5 Bisphosphate Dic8, 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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    4 5 bisphosphate  (Echelon Biosciences)


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    Echelon Biosciences 4 5 bisphosphate
    4 5 Bisphosphate, 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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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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  • 86
    Echelon Biosciences phosphatidylinositol 4 5 bisphosphate
    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).
    Phosphatidylinositol 4 5 Bisphosphate, 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
    https://www.bioz.com/result/phosphatidylinositol 4 5 bisphosphate/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphatidylinositol 4 5 bisphosphate - by Bioz Stars, 2024-03
    86/100 stars
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    86
    Echelon Biosciences d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl
    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).
    D Myo Phosphatidylinositol 4 5 Bisphosphate Ptdins 4 5 P2 D Sn 1 2 Di O Octanoylglyceryl, 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
    https://www.bioz.com/result/d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl - by Bioz Stars, 2024-03
    86/100 stars
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    86
    Echelon Biosciences phosphoinositol 4 5 bisphosphate pip2
    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).
    Phosphoinositol 4 5 Bisphosphate Pip2, 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
    https://www.bioz.com/result/phosphoinositol 4 5 bisphosphate pip2/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphoinositol 4 5 bisphosphate pip2 - by Bioz Stars, 2024-03
    86/100 stars
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    86
    Echelon Biosciences 4 5 bisphosphate
    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).
    4 5 Bisphosphate, 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
    https://www.bioz.com/result/4 5 bisphosphate/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    4 5 bisphosphate - by Bioz Stars, 2024-03
    86/100 stars
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    86
    Echelon Biosciences l α phosphatidylinositol 4 5 bisphosphate
    (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).
    L α Phosphatidylinositol 4 5 Bisphosphate, 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
    https://www.bioz.com/result/l α phosphatidylinositol 4 5 bisphosphate/product/Echelon Biosciences
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    l α phosphatidylinositol 4 5 bisphosphate - by Bioz Stars, 2024-03
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    Echelon Biosciences 4 5 bisphosphate dic8
    (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).
    4 5 Bisphosphate Dic8, 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
    https://www.bioz.com/result/4 5 bisphosphate dic8/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    4 5 bisphosphate dic8 - by Bioz Stars, 2024-03
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    Image Search Results


    PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).

    Journal: bioRxiv

    Article Title: PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway

    doi: 10.1101/2024.02.23.581751

    Figure Lengend Snippet: PTEN and CnrN cause differential effects on PI(4,5)P 2 and PI(3,4,5)P 3 accumulation during chemorepulsion. (A-L) Cells were incubated in HL5 with 300 ng/ml AprA or an equivalent volume of buffer for the indicated amounts of time. Phosphatidylinositol were extracted and quantified using PI(4,5)P 2 (A, and C-G) and PI(3,4,5)P 3 (B, and H-L) ELISAs. Values in (A) and (B) are the 0 seconds values from (C - L). Values are mean ± SEM from 4 (C, J, and L), 5 (D, E, G, I, and K), or 6 (F, H) independent experiments. * p L 0.05, ** p L 0.01, *** p L 0.001, **** p L 0.0001 compared to the WT (A and B) or the 0 time point (C-L), unless otherwise indicated (A and B, One-way ANOVA with Tukey’s correction) (C-L, Mann-Whitney U test).

    Article Snippet: Phosphatidylinositol extractions and ELISAs were performed following the manufacturer’s directions for the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) Mass ELISA kit (# K-4500) and the phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) Mass ELISA kit (#K-2500s, Echelon Biosciences Inc, Salt Lake City, UT).

    Techniques: Incubation, MANN-WHITNEY

    (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).

    Journal: bioRxiv

    Article Title: Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback

    doi: 10.1101/2023.09.04.556152

    Figure Lengend Snippet: (A) Cartoon illustrating how the W365P mutation disrupts the specificity loop alpha helical conformation in PIP5KB. (B) Lipid kinase activity measured in the presence of PIP5KB and PIP5KB (W365P). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ and the indicated concentration of PIP5KB. (C) PIP5KB (WT and D51R) but not PIP5KB (W365P) display cooperative PI(4,5) P 2 dependent changes in supported membrane localization. Phosphorylation of PI(4)P and membrane localization of AF647-PIP5KB were monitored in the presence of 5 nM AF647-PIP5KB (WT or mutant) and 20 nM Cy3-PLCδ. (B-C) Initial membrane composition: 96% DOPC, 4% PI(4)P. (D) Representative TIRF-M images showing membrane localization in the presence of 100 pM AF647-PIP5KB or AF647-PIP5KB (W365P). (E) Experimental measure of membrane binding frequency ( k ON ) in the presence of 10-50 pM AF647-PIP5KB and 20-100 pM AF647-PIP5KB (W365P). (F-G) Single molecule dwell time distributions measured in the presence of either AF647-5KB or AF647-5KB(W365P). (H) Step size distribution comparing the membrane diffusivity of AF647-5KB and AF647-5KB(W365P). (D-H) Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (I) Representative TIRF-M images of mEos3.2-PIP5KB and mEos3.2-PIP5KB(W365P) plasma membrane localization following photoconversion with 405 nm light in HEK293T cells. (J) Single molecule dwell time distributions of mEos3.2-PIP5KB and mEos3.2-PIP5KB (W365P).

    Article Snippet: The following lipids were used to generated small unilamellar vesicles (SUVs): 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 DOPC, Avanti # 850375C), L-α-phosphatidylinositol-4-phosphate (Brain PI(4)P, Avanti Cat# 840045X), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-5’-phosphate) (PI(5)P, Avanti Cat# 850152P), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P 2 , Avanti # 840046X), D-myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16, Echelon P-3916-100ug), D-myo-phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 diC16, Echelon P-3416-100ug), 1,2-dioleoyl- sn- glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE, Avanti #780201C).

    Techniques: Mutagenesis, Activity Assay, Concentration Assay, Membrane, Binding Assay

    (A) Cartoon showing the structure of the mNG-PIP5K specificity loop-PLCδ chimeric protein. The expected PIP lipid binding interactions are indicated. (B) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing either 4% PI(4)P or 4% PI(5)P. (C) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing 4% PI(4,5)P 2 . (D) Sequence alignment of PIP5K and PIP4K specificity loops. (E) Cartoon showing the PIP4K to PIP5K specificity loop swap. (F) Quantification of the fold increase in localization of Cy5-PIP4K and Cy5-PIP4K(5K-SL) on membranes containing different molar concentrations of PI(4,5)P 2 . The fold change was calculated relative to the measured membrane density in the absence of PI(4,5)P 2 . (G) Single molecule dwell time distributions of Cy5-PIP4K and Cy5-PIP4K(5K-SL) measured on membranes containing 4% PI(4,5)P 2 . (H) Plots showing the cumulative membrane binding events measured in the presence of 20-80 pM Cy5-PIP4K. The slope of each curve was calculated by linear regression yielding a binding frequency for each concentration of Cy5-PIP4K. (I) Experimental measure of membrane binding frequency ( k ON ) in the presence of 20-80 pM Cy5-PIP4K or 5-10 pM Cy5-PIP4K(5K-SL).

    Journal: bioRxiv

    Article Title: Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback

    doi: 10.1101/2023.09.04.556152

    Figure Lengend Snippet: (A) Cartoon showing the structure of the mNG-PIP5K specificity loop-PLCδ chimeric protein. The expected PIP lipid binding interactions are indicated. (B) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing either 4% PI(4)P or 4% PI(5)P. (C) Single molecule dwell time distributions of mNG-PLCδ and mNG-(5K-SL)-PLCδ measured on membranes containing 4% PI(4,5)P 2 . (D) Sequence alignment of PIP5K and PIP4K specificity loops. (E) Cartoon showing the PIP4K to PIP5K specificity loop swap. (F) Quantification of the fold increase in localization of Cy5-PIP4K and Cy5-PIP4K(5K-SL) on membranes containing different molar concentrations of PI(4,5)P 2 . The fold change was calculated relative to the measured membrane density in the absence of PI(4,5)P 2 . (G) Single molecule dwell time distributions of Cy5-PIP4K and Cy5-PIP4K(5K-SL) measured on membranes containing 4% PI(4,5)P 2 . (H) Plots showing the cumulative membrane binding events measured in the presence of 20-80 pM Cy5-PIP4K. The slope of each curve was calculated by linear regression yielding a binding frequency for each concentration of Cy5-PIP4K. (I) Experimental measure of membrane binding frequency ( k ON ) in the presence of 20-80 pM Cy5-PIP4K or 5-10 pM Cy5-PIP4K(5K-SL).

    Article Snippet: The following lipids were used to generated small unilamellar vesicles (SUVs): 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 DOPC, Avanti # 850375C), L-α-phosphatidylinositol-4-phosphate (Brain PI(4)P, Avanti Cat# 840045X), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-5’-phosphate) (PI(5)P, Avanti Cat# 850152P), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P 2 , Avanti # 840046X), D-myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16, Echelon P-3916-100ug), D-myo-phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 diC16, Echelon P-3416-100ug), 1,2-dioleoyl- sn- glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE, Avanti #780201C).

    Techniques: Binding Assay, Sequencing, Membrane, Concentration Assay

    (A) AlphaFold structure of human PIP5KB validated against zPIP5Kα (5E3U.pdb). Structure highlights the specificity loop (red) and the PIP lipid (or substrate) Binding Motif (PIPBM, blue). (B) Single molecule dwell time distributions of AF647-PIP5KB measured in the buffer containing either 1 mM ATP or ADP. (C) Bulk membrane recruitment of either 25nM mNG-PIP5KB or mNG-PIP5KB (K205A/ R211A). (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Single molecule dwell time distribution comparing mNG-PIP5KB and mNG-PIP5KB (K205A/R211A). Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (E) Representative TIRF-M images showing the localization of the mEos3.2-PIP5KB (WT and K205A/R211A) expressed in HEK293T cells. (F) Kymograph showing the single molecule plasma membrane binding dynamics of mEos3.2-PIP5KB (WT and K205A/R211A) in HEK293T cells. (G) Single molecule dwell time distributions for mEos3.2-PIP5KB (WT and K205A/R211A) measured in HEK293T cells. (H) Schematic showing SpyTag-PIP5KB(K205A/ R211A) and SpyCatcher kinase activity assay. (I) Kinetics of lipid phosphorylation measured for membrane tethered and non-tethered SpyTag-PIP5KB(K205A/R211A). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ.

    Journal: bioRxiv

    Article Title: Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback

    doi: 10.1101/2023.09.04.556152

    Figure Lengend Snippet: (A) AlphaFold structure of human PIP5KB validated against zPIP5Kα (5E3U.pdb). Structure highlights the specificity loop (red) and the PIP lipid (or substrate) Binding Motif (PIPBM, blue). (B) Single molecule dwell time distributions of AF647-PIP5KB measured in the buffer containing either 1 mM ATP or ADP. (C) Bulk membrane recruitment of either 25nM mNG-PIP5KB or mNG-PIP5KB (K205A/ R211A). (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Single molecule dwell time distribution comparing mNG-PIP5KB and mNG-PIP5KB (K205A/R211A). Membrane composition: 96% DOPC, 4% PI(4,5)P 2 . (E) Representative TIRF-M images showing the localization of the mEos3.2-PIP5KB (WT and K205A/R211A) expressed in HEK293T cells. (F) Kymograph showing the single molecule plasma membrane binding dynamics of mEos3.2-PIP5KB (WT and K205A/R211A) in HEK293T cells. (G) Single molecule dwell time distributions for mEos3.2-PIP5KB (WT and K205A/R211A) measured in HEK293T cells. (H) Schematic showing SpyTag-PIP5KB(K205A/ R211A) and SpyCatcher kinase activity assay. (I) Kinetics of lipid phosphorylation measured for membrane tethered and non-tethered SpyTag-PIP5KB(K205A/R211A). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ.

    Article Snippet: The following lipids were used to generated small unilamellar vesicles (SUVs): 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 DOPC, Avanti # 850375C), L-α-phosphatidylinositol-4-phosphate (Brain PI(4)P, Avanti Cat# 840045X), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-5’-phosphate) (PI(5)P, Avanti Cat# 850152P), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P 2 , Avanti # 840046X), D-myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16, Echelon P-3916-100ug), D-myo-phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 diC16, Echelon P-3416-100ug), 1,2-dioleoyl- sn- glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE, Avanti #780201C).

    Techniques: Binding Assay, Membrane, Kinase Assay

    (A) Single molecule dwell time distributions measured in the presence of AF647-PIP5KB on SLBs containing 2% of the indicated PIP lipid. (B) Kinetics of PIP5KB dependent lipid phosphorylation measured in the presence of 0-20% DOPS. All membranes contain 2% PI(4)P and reactions were monitored using 20 nM Cy3-PLCδ. Kinetics were quantified by comparing the half-time for reaction completion. (C) Quantification of the fold increase of membrane recruitment measured in the presence of 15 nM mNG-PIP5KB on SLBs containing 0-20% DOPS. The fold increase in membrane binding we calculated relative to the membrane intensity of mNG-PIP5K measured in the absence of DOPS. (D) Single molecule dwell time analysis of AF647-PIP5KB in the presence of 20% DOPS or 2% PI(4,5)P 2 . (E) Representative TIRF-M images showing the membrane localization of 15 nM mNG-PIP5K or mNG-PIP5K (K205A/R211A). Membrane composition: 20% DOPS, 80% DOPC. (F) Quantification of bulk recruitment data shown in (E).

    Journal: bioRxiv

    Article Title: Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback

    doi: 10.1101/2023.09.04.556152

    Figure Lengend Snippet: (A) Single molecule dwell time distributions measured in the presence of AF647-PIP5KB on SLBs containing 2% of the indicated PIP lipid. (B) Kinetics of PIP5KB dependent lipid phosphorylation measured in the presence of 0-20% DOPS. All membranes contain 2% PI(4)P and reactions were monitored using 20 nM Cy3-PLCδ. Kinetics were quantified by comparing the half-time for reaction completion. (C) Quantification of the fold increase of membrane recruitment measured in the presence of 15 nM mNG-PIP5KB on SLBs containing 0-20% DOPS. The fold increase in membrane binding we calculated relative to the membrane intensity of mNG-PIP5K measured in the absence of DOPS. (D) Single molecule dwell time analysis of AF647-PIP5KB in the presence of 20% DOPS or 2% PI(4,5)P 2 . (E) Representative TIRF-M images showing the membrane localization of 15 nM mNG-PIP5K or mNG-PIP5K (K205A/R211A). Membrane composition: 20% DOPS, 80% DOPC. (F) Quantification of bulk recruitment data shown in (E).

    Article Snippet: The following lipids were used to generated small unilamellar vesicles (SUVs): 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 DOPC, Avanti # 850375C), L-α-phosphatidylinositol-4-phosphate (Brain PI(4)P, Avanti Cat# 840045X), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-5’-phosphate) (PI(5)P, Avanti Cat# 850152P), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P 2 , Avanti # 840046X), D-myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16, Echelon P-3916-100ug), D-myo-phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 diC16, Echelon P-3416-100ug), 1,2-dioleoyl- sn- glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE, Avanti #780201C).

    Techniques: Membrane, Binding Assay

    (A) Structure of PIP5K (5E3U.pdb) showing the position of residues that are important for catalysis. (B) Representative TIRF-M images showing the localization of mNG-PIP5KB (WT and mutants). (C) Single molecule dwell time distributions for mNG-PIP5KB (WT and mutants) measured. (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Bulk membrane recruitment measured by TIRF-M in the presence of 20 nM mNG-PIP5KB (WT and mutants). Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (E-F) Lipid kinase activity measured using TIRF-M in the presence of 10 nM PIP5KB (WT and mutants). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ. (E) Membrane composition: 96% DOPC, 4% PI(4)P. (F) Membrane composition: 78% DOPC, 2% PI(4)P, 20% DOPS.

    Journal: bioRxiv

    Article Title: Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P 2 with positive feedback

    doi: 10.1101/2023.09.04.556152

    Figure Lengend Snippet: (A) Structure of PIP5K (5E3U.pdb) showing the position of residues that are important for catalysis. (B) Representative TIRF-M images showing the localization of mNG-PIP5KB (WT and mutants). (C) Single molecule dwell time distributions for mNG-PIP5KB (WT and mutants) measured. (B-C) Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (D) Bulk membrane recruitment measured by TIRF-M in the presence of 20 nM mNG-PIP5KB (WT and mutants). Membrane composition: 98% DOPC, 2% PI(4,5)P 2 . (E-F) Lipid kinase activity measured using TIRF-M in the presence of 10 nM PIP5KB (WT and mutants). Production of PI(4,5)P 2 was monitored in the presence of 20 nM Cy3-PLCδ. (E) Membrane composition: 96% DOPC, 4% PI(4)P. (F) Membrane composition: 78% DOPC, 2% PI(4)P, 20% DOPS.

    Article Snippet: The following lipids were used to generated small unilamellar vesicles (SUVs): 1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1 DOPC, Avanti # 850375C), L-α-phosphatidylinositol-4-phosphate (Brain PI(4)P, Avanti Cat# 840045X), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-myo-inositol-5’-phosphate) (PI(5)P, Avanti Cat# 850152P), L-α-phosphatidylinositol-4,5-bisphosphate (Brain PI(4,5)P 2 , Avanti # 840046X), D-myo-phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 diC16, Echelon P-3916-100ug), D-myo-phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 diC16, Echelon P-3416-100ug), 1,2-dioleoyl- sn- glycero-3-phospho-L-serine (18:1 DOPS, Avanti # 840035C), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC-PE, Avanti #780201C).

    Techniques: Membrane, Activity Assay