p-3016  (Echelon Biosciences)


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    Echelon Biosciences p-3016
    P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p-3016  (Echelon Biosciences)


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    Echelon Biosciences p-3016
    P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phosphatidylinositol 3 phosphate dic16  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16
    ( A to F ) Titration series of H3K9me3 peptide (in the absence and presence of di-C 16:0 PI5P), PI5P, or <t>PI3P</t> with fluorescently labeled, mutant, and wild-type, full-length hUHRF1 were analyzed by MST. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; and D142R649*: D142A, R649A. n = 3; error bars: SD; K d values are listed in tables S1 and S2.
    Phosphatidylinositol 3 Phosphate Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P"

    Article Title: Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P

    Journal: Science Advances

    doi: 10.1126/sciadv.abl9461

    ( A to F ) Titration series of H3K9me3 peptide (in the absence and presence of di-C 16:0 PI5P), PI5P, or PI3P with fluorescently labeled, mutant, and wild-type, full-length hUHRF1 were analyzed by MST. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; and D142R649*: D142A, R649A. n = 3; error bars: SD; K d values are listed in tables S1 and S2.
    Figure Legend Snippet: ( A to F ) Titration series of H3K9me3 peptide (in the absence and presence of di-C 16:0 PI5P), PI5P, or PI3P with fluorescently labeled, mutant, and wild-type, full-length hUHRF1 were analyzed by MST. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; and D142R649*: D142A, R649A. n = 3; error bars: SD; K d values are listed in tables S1 and S2.

    Techniques Used: Titration, Labeling, Mutagenesis

    p-3016  (Echelon Biosciences)


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    Echelon Biosciences p-3016
    P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p-3016  (Echelon Biosciences)


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    Echelon Biosciences p-3016
    P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p-3016  (Echelon Biosciences)


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    Echelon Biosciences p-3016
    P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phosphatidylinositol 3 phosphate dic16 pi3p  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16 pi3p
    Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− <t>phosphatidylinositol-3</t> phosphatase <t>(PI3P)</t> (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.
    Phosphatidylinositol 3 Phosphate Dic16 Pi3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients"

    Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2021.742862

    Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.
    Figure Legend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

    Techniques Used: Inhibition, Patch Clamp, Transferring, Fluorescence, Flow Cytometry

    phosphatidylinositol 3 phosphate dic16  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16
    a , The consensus at each amino acid position of the percentual difference of HDX at each time point is plotted with and without PI5P for each of >200 unique partially overlapping peptides of hUHRF1. The color key for binning of HDX differences is shown at the right. White regions indicate gaps in the obtained peptide coverage of hUHRF1. Roman numerals indicate regions highlighted in the PDB structure in (f). Scheme illustrating domain architecture of hUHRF1 is at the top. b to e , HDX time curves of specific peptides encompassing the TTD surface groove (b), the Linker 2 (c), the N-terminal H3 tail-interacting residues of the PHD (d) and the DNA binding region of the SRA (e) are shown. Each experiment was performed in triplicate; asterisks indicate time points with P < 0.01 (Student’s t test between hUHRF1(PI5P) and <t>hUHRF1(PI3P)</t> experiments). Dashed line reflects full deuteration level of the corresponding peptide. Error bars represent SD from three independent measurements. f , Combined model of the crystal structure of the TTD-PHD module (4GY5) and the SRA domain (3CLZ) of hUHRF1, highlighting regions of interest and colored by domain. The numbered regions highlighted in blue (I, II and III) are indicating regions of slow HDX. Region IV highlighted in red shows fast HDX. Region (V) is highlighted in blue based on slow HDX as shown in Extended Data Fig. 6. The dotted lines point out the arbitrary continuation of the structure. g , Titration series of di-C 16:0 PI5P with fluorescently labeled protein domains (TTD and PHD) and Linker 2 of hUHRF1 analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 1. h , Titration series of wild type and mutated Linker 2 with fluorescently labeled wild type and mutated Linker 4 were analyzed in absence and presence of di-C 16:0 PI5P or PI3P by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 1.
    Phosphatidylinositol 3 Phosphate Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Molecular basis of UHRF1 allosteric activation for synergistic histone modification binding by PI5P"

    Article Title: Molecular basis of UHRF1 allosteric activation for synergistic histone modification binding by PI5P

    Journal: bioRxiv

    doi: 10.1101/2021.08.04.455045

    a , The consensus at each amino acid position of the percentual difference of HDX at each time point is plotted with and without PI5P for each of >200 unique partially overlapping peptides of hUHRF1. The color key for binning of HDX differences is shown at the right. White regions indicate gaps in the obtained peptide coverage of hUHRF1. Roman numerals indicate regions highlighted in the PDB structure in (f). Scheme illustrating domain architecture of hUHRF1 is at the top. b to e , HDX time curves of specific peptides encompassing the TTD surface groove (b), the Linker 2 (c), the N-terminal H3 tail-interacting residues of the PHD (d) and the DNA binding region of the SRA (e) are shown. Each experiment was performed in triplicate; asterisks indicate time points with P < 0.01 (Student’s t test between hUHRF1(PI5P) and hUHRF1(PI3P) experiments). Dashed line reflects full deuteration level of the corresponding peptide. Error bars represent SD from three independent measurements. f , Combined model of the crystal structure of the TTD-PHD module (4GY5) and the SRA domain (3CLZ) of hUHRF1, highlighting regions of interest and colored by domain. The numbered regions highlighted in blue (I, II and III) are indicating regions of slow HDX. Region IV highlighted in red shows fast HDX. Region (V) is highlighted in blue based on slow HDX as shown in Extended Data Fig. 6. The dotted lines point out the arbitrary continuation of the structure. g , Titration series of di-C 16:0 PI5P with fluorescently labeled protein domains (TTD and PHD) and Linker 2 of hUHRF1 analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 1. h , Titration series of wild type and mutated Linker 2 with fluorescently labeled wild type and mutated Linker 4 were analyzed in absence and presence of di-C 16:0 PI5P or PI3P by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 1.
    Figure Legend Snippet: a , The consensus at each amino acid position of the percentual difference of HDX at each time point is plotted with and without PI5P for each of >200 unique partially overlapping peptides of hUHRF1. The color key for binning of HDX differences is shown at the right. White regions indicate gaps in the obtained peptide coverage of hUHRF1. Roman numerals indicate regions highlighted in the PDB structure in (f). Scheme illustrating domain architecture of hUHRF1 is at the top. b to e , HDX time curves of specific peptides encompassing the TTD surface groove (b), the Linker 2 (c), the N-terminal H3 tail-interacting residues of the PHD (d) and the DNA binding region of the SRA (e) are shown. Each experiment was performed in triplicate; asterisks indicate time points with P < 0.01 (Student’s t test between hUHRF1(PI5P) and hUHRF1(PI3P) experiments). Dashed line reflects full deuteration level of the corresponding peptide. Error bars represent SD from three independent measurements. f , Combined model of the crystal structure of the TTD-PHD module (4GY5) and the SRA domain (3CLZ) of hUHRF1, highlighting regions of interest and colored by domain. The numbered regions highlighted in blue (I, II and III) are indicating regions of slow HDX. Region IV highlighted in red shows fast HDX. Region (V) is highlighted in blue based on slow HDX as shown in Extended Data Fig. 6. The dotted lines point out the arbitrary continuation of the structure. g , Titration series of di-C 16:0 PI5P with fluorescently labeled protein domains (TTD and PHD) and Linker 2 of hUHRF1 analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 1. h , Titration series of wild type and mutated Linker 2 with fluorescently labeled wild type and mutated Linker 4 were analyzed in absence and presence of di-C 16:0 PI5P or PI3P by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 1.

    Techniques Used: Binding Assay, Titration, Labeling, Microscale Thermophoresis

    a to d , Titration series of H3K9me3 peptide with fluorescently labeled, mutant, full-length hUHRF1 in absence and presence of di-C 16:0 PI5P were analyzed by microscale thermophoresis. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; D142R649*: D142A, R649A. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values of all measurements are listed in Extended Data Table 1. e , Titration series of di-C 16:0 PI5P with fluorescently labeled hUHRF1 carrying the indicated mutations were analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 2. f , Titration series of di-C 16:0 PI3P with fluorescently labeled, WT and mutant, full-length hUHRF1 were analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 2. g , Putative conformational states of wild type and mutant hUHRF1 proteins. (i) In the apo/ ground state of wild type hUHRF1, the TTD domain is blocked by PBR/ Linker 4. Only the PHD domain is accessible for H3 N-terminal tail binding. (ii) PI5P recognizes and stabilizes a particular conformational state of hUHRF1. In this activated state, Linker 4 is displaced from the TTD surface; Linker 2 is positioned on the TTD surface groove and bridged to Linker 4 by PI5P. This conformational transition locks the TTD and PHD in a particular three-dimensional state that promotes multivalent, synergistic recognition of the H3K9me3 tail. In hUHRF1 PBR*, Linker 4 cannot block the TTD surface. Linker 2 remains dynamic and flexible allowing both compact (iii) and extended (iv) conformations. Despite free accessibility, it is not permanently bound to the TTD surface groove. In hUHRF1 R649* the TTD is unblocked. Like the wild type protein, it can interact with PI5P and form the activated conformational state for enhanced H3K9me3 recognition (ii). hUHRF1 R296* binds to PI5P only via the PBR/ Linker 4. While this releases the PBR-mediated TTD blocking, the mutant Linker 2 cannot be stably positioned on the TTD surface groove. In consequence hUHRF1 R296* is unable to adopt the activated conformation required for multivalent, synergistic H3K9me3 tail binding (iv). The conformational states of hUHRF1 R296R649* and hUHRF1 D142R649* are similar to that of hUHRF1 PBR* with the difference that PI5P is bound to the PBR. Linker 4 is displaced from the surface of the TTD but due to the mutations in the TTD surface groove or Linker 2 these cannot interact.
    Figure Legend Snippet: a to d , Titration series of H3K9me3 peptide with fluorescently labeled, mutant, full-length hUHRF1 in absence and presence of di-C 16:0 PI5P were analyzed by microscale thermophoresis. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; D142R649*: D142A, R649A. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values of all measurements are listed in Extended Data Table 1. e , Titration series of di-C 16:0 PI5P with fluorescently labeled hUHRF1 carrying the indicated mutations were analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 2. f , Titration series of di-C 16:0 PI3P with fluorescently labeled, WT and mutant, full-length hUHRF1 were analyzed by microscale thermophoresis. Data are plotted as average of three independent experiments; error bars correspond to SD. K D values are listed in Extended Data Table 2. g , Putative conformational states of wild type and mutant hUHRF1 proteins. (i) In the apo/ ground state of wild type hUHRF1, the TTD domain is blocked by PBR/ Linker 4. Only the PHD domain is accessible for H3 N-terminal tail binding. (ii) PI5P recognizes and stabilizes a particular conformational state of hUHRF1. In this activated state, Linker 4 is displaced from the TTD surface; Linker 2 is positioned on the TTD surface groove and bridged to Linker 4 by PI5P. This conformational transition locks the TTD and PHD in a particular three-dimensional state that promotes multivalent, synergistic recognition of the H3K9me3 tail. In hUHRF1 PBR*, Linker 4 cannot block the TTD surface. Linker 2 remains dynamic and flexible allowing both compact (iii) and extended (iv) conformations. Despite free accessibility, it is not permanently bound to the TTD surface groove. In hUHRF1 R649* the TTD is unblocked. Like the wild type protein, it can interact with PI5P and form the activated conformational state for enhanced H3K9me3 recognition (ii). hUHRF1 R296* binds to PI5P only via the PBR/ Linker 4. While this releases the PBR-mediated TTD blocking, the mutant Linker 2 cannot be stably positioned on the TTD surface groove. In consequence hUHRF1 R296* is unable to adopt the activated conformation required for multivalent, synergistic H3K9me3 tail binding (iv). The conformational states of hUHRF1 R296R649* and hUHRF1 D142R649* are similar to that of hUHRF1 PBR* with the difference that PI5P is bound to the PBR. Linker 4 is displaced from the surface of the TTD but due to the mutations in the TTD surface groove or Linker 2 these cannot interact.

    Techniques Used: Titration, Labeling, Mutagenesis, Microscale Thermophoresis, Binding Assay, Blocking Assay, Stable Transfection

    phosphatidylinositol 3 phosphate dic16  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16
    The BCAS3-C16orf70 complex binds to <t>PtdIns3P</t> in vitro . (A) CBB staining of purified GFP-His, GFP-WIPI1-His, GFP-BCAS3-His/GFP-C16orf70-His and GFP-BCAS3 R401A -His/GFP-C16orf70-His. (B) Schematic diagram of the liposome flotation assay. After incubating the liposome solution with purified proteins, OptiPrep was added to a final concentration of 35% and then 30% OptiPrep and buffer were sequentially layered on top. After centrifugation, the Bottom (B), Middle (M) and Top (T) fractions are collected and analyzed by western blotting. (C) Flotation assays using the purified proteins and liposomes containing the indicated phosphoinositides were performed. GFP-His and GFP-WIPI1-His were detected with an anti-GFP antibody, and GFP-BCAS3-His was detected with an anti-BCAS3 antibody. (D) Flotation assays using liposomes with various concentrations of PtdIns3P were performed. GFP-BCAS3-His was detected with an anti-BCAS3 antibody
    Phosphatidylinositol 3 Phosphate Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phosphatidylinositol 3 phosphate dic16 - by Bioz Stars, 2024-05
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    Images

    1) Product Images from "Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy"

    Article Title: Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy

    Journal: Autophagy

    doi: 10.1080/15548627.2021.1874133

    The BCAS3-C16orf70 complex binds to PtdIns3P in vitro . (A) CBB staining of purified GFP-His, GFP-WIPI1-His, GFP-BCAS3-His/GFP-C16orf70-His and GFP-BCAS3 R401A -His/GFP-C16orf70-His. (B) Schematic diagram of the liposome flotation assay. After incubating the liposome solution with purified proteins, OptiPrep was added to a final concentration of 35% and then 30% OptiPrep and buffer were sequentially layered on top. After centrifugation, the Bottom (B), Middle (M) and Top (T) fractions are collected and analyzed by western blotting. (C) Flotation assays using the purified proteins and liposomes containing the indicated phosphoinositides were performed. GFP-His and GFP-WIPI1-His were detected with an anti-GFP antibody, and GFP-BCAS3-His was detected with an anti-BCAS3 antibody. (D) Flotation assays using liposomes with various concentrations of PtdIns3P were performed. GFP-BCAS3-His was detected with an anti-BCAS3 antibody
    Figure Legend Snippet: The BCAS3-C16orf70 complex binds to PtdIns3P in vitro . (A) CBB staining of purified GFP-His, GFP-WIPI1-His, GFP-BCAS3-His/GFP-C16orf70-His and GFP-BCAS3 R401A -His/GFP-C16orf70-His. (B) Schematic diagram of the liposome flotation assay. After incubating the liposome solution with purified proteins, OptiPrep was added to a final concentration of 35% and then 30% OptiPrep and buffer were sequentially layered on top. After centrifugation, the Bottom (B), Middle (M) and Top (T) fractions are collected and analyzed by western blotting. (C) Flotation assays using the purified proteins and liposomes containing the indicated phosphoinositides were performed. GFP-His and GFP-WIPI1-His were detected with an anti-GFP antibody, and GFP-BCAS3-His was detected with an anti-BCAS3 antibody. (D) Flotation assays using liposomes with various concentrations of PtdIns3P were performed. GFP-BCAS3-His was detected with an anti-BCAS3 antibody

    Techniques Used: In Vitro, Staining, Purification, Concentration Assay, Centrifugation, Western Blot

    phosphatidylinositol 3 phosphate dic16 pi 3 p dic16  (Echelon Biosciences)


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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16 pi 3 p dic16
    MCBs with PC/PS and EE lipid composition containing 1 mol% <t>PI(3)P</t> ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean equatorial GFP signal intensity in A–D ). (p=<0.0001; n = 20) ( F ) MCBs with PC/PS and PC/PS/CH lipid composition (0 mol% PI(3)P) incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Graph presents mean equatorial GFP signal intensity (p=0.005; n = 25). For both E ) and F ) GFP signal intensity is normalized to DiD signal intensity, however the same pattern can be seen in the raw intensity values.
    Phosphatidylinositol 3 Phosphate Dic16 Pi 3 P Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "A non-linear system patterns Rab5 GTPase on the membrane"

    Article Title: A non-linear system patterns Rab5 GTPase on the membrane

    Journal: eLife

    doi: 10.7554/eLife.54434

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean equatorial GFP signal intensity in A–D ). (p=<0.0001; n = 20) ( F ) MCBs with PC/PS and PC/PS/CH lipid composition (0 mol% PI(3)P) incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Graph presents mean equatorial GFP signal intensity (p=0.005; n = 25). For both E ) and F ) GFP signal intensity is normalized to DiD signal intensity, however the same pattern can be seen in the raw intensity values.
    Figure Legend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean equatorial GFP signal intensity in A–D ). (p=<0.0001; n = 20) ( F ) MCBs with PC/PS and PC/PS/CH lipid composition (0 mol% PI(3)P) incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Graph presents mean equatorial GFP signal intensity (p=0.005; n = 25). For both E ) and F ) GFP signal intensity is normalized to DiD signal intensity, however the same pattern can be seen in the raw intensity values.

    Techniques Used: Incubation

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.
    Figure Legend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.

    Techniques Used: Incubation

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean GFP-Rab5 signal intensity outside of and within segmented domains in B ) and D ) (p=<0.0001) (See also ). ( F ) Mean GFP-Rab5 signal intensity outside of and within segmented domains on MCBs with PC/PS/CH/PlasmPE and PC/PS/CH/SM lipid composition containing 1 mol% PI(3)P (p=<0.0046) (See also ).
    Figure Legend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean GFP-Rab5 signal intensity outside of and within segmented domains in B ) and D ) (p=<0.0001) (See also ). ( F ) Mean GFP-Rab5 signal intensity outside of and within segmented domains on MCBs with PC/PS/CH/PlasmPE and PC/PS/CH/SM lipid composition containing 1 mol% PI(3)P (p=<0.0046) (See also ).

    Techniques Used: Incubation

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.
    Figure Legend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.

    Techniques Used: Incubation


    Figure Legend Snippet:

    Techniques Used: Recombinant, Expressing, Plasmid Preparation, Ligation, Transfection, Construct, Microscopy, BIA-KA

    p-3016  (Echelon Biosciences)


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    Echelon Biosciences p-3016
    P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences p-3016
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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16
    ( A to F ) Titration series of H3K9me3 peptide (in the absence and presence of di-C 16:0 PI5P), PI5P, or <t>PI3P</t> with fluorescently labeled, mutant, and wild-type, full-length hUHRF1 were analyzed by MST. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; and D142R649*: D142A, R649A. n = 3; error bars: SD; K d values are listed in tables S1 and S2.
    Phosphatidylinositol 3 Phosphate Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16 pi3p
    Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− <t>phosphatidylinositol-3</t> phosphatase <t>(PI3P)</t> (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.
    Phosphatidylinositol 3 Phosphate Dic16 Pi3p, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences phosphatidylinositol 3 phosphate dic16 pi 3 p dic16
    MCBs with PC/PS and EE lipid composition containing 1 mol% <t>PI(3)P</t> ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean equatorial GFP signal intensity in A–D ). (p=<0.0001; n = 20) ( F ) MCBs with PC/PS and PC/PS/CH lipid composition (0 mol% PI(3)P) incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Graph presents mean equatorial GFP signal intensity (p=0.005; n = 25). For both E ) and F ) GFP signal intensity is normalized to DiD signal intensity, however the same pattern can be seen in the raw intensity values.
    Phosphatidylinositol 3 Phosphate Dic16 Pi 3 P Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ( A to F ) Titration series of H3K9me3 peptide (in the absence and presence of di-C 16:0 PI5P), PI5P, or PI3P with fluorescently labeled, mutant, and wild-type, full-length hUHRF1 were analyzed by MST. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; and D142R649*: D142A, R649A. n = 3; error bars: SD; K d values are listed in tables S1 and S2.

    Journal: Science Advances

    Article Title: Molecular basis of hUHRF1 allosteric activation for synergistic histone modification binding by PI5P

    doi: 10.1126/sciadv.abl9461

    Figure Lengend Snippet: ( A to F ) Titration series of H3K9me3 peptide (in the absence and presence of di-C 16:0 PI5P), PI5P, or PI3P with fluorescently labeled, mutant, and wild-type, full-length hUHRF1 were analyzed by MST. PBR*: K644A, K646A, K648A, R649A, K650A, S651A; R296*: R296A; R649*: R649A; D142*: D142A; R296R649*: R296A, R649A; and D142R649*: D142A, R649A. n = 3; error bars: SD; K d values are listed in tables S1 and S2.

    Article Snippet: Phosphatidylinositol 5-phosphate diC16 (di-C 16:0 PI5P; Echelon, #P5016), phosphatidylinositol 4-phosphate diC16 (di-C 16:0 PI4P; Echelon, #P4016), phosphatidylinositol 3-phosphate diC16 (di-C 16:0 PI3P; Echelon, #P3016), phosphatidylinositol 4,5-bisphosphate diC16 [di-C 16:0 PI(4,5)P2; Echelon, #P4516], phosphatidylinositol 3,5-bisphosphate diC16 [di-C 16:0 PI(3,5)P2; Echelon, #P3516], phosphatidylinositol 5-phosphate diC8 (di-C 8:0 PI5P; Echelon, #P-5008), inositol 1,5-bisphosphate [Ins(1,5)P2; Echelon, #Q-0015], 1,2-dioleoyl- sn -glycero-3-phospho-(1′-myo-inositol-5′-phosphate) (di-C 18:1 PI5P; Avanti Polar Lipids, #850152P), 1-heptadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)- sn -glycero-3-phospho-(1′-myo-inositol-5′-phosphate) (17:0,20:4 PI5P; Avanti Polar Lipids, #LM1902), 1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine- N -(biotinyl) (sodium salt) (di-C 16:0 biotinyl PE; Avanti Polar Lipids, #870285), 1,2-dimyristoyl- sn -glycero-3-phosphoethanolamine (di-C 14:0 PE; Avanti Polar Lipids, #850745), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine- N -(biotinyl) (sodium salt) (di-C 18:1 biotinyl PE; Avanti Polar Lipids, #870282), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[methoxy(polyethylene glycol)-550] (ammonium salt) (di-C 18:0 PEG550PE; Avanti Polar Lipids, #880520), cholesterol-(polyethylene glycol-600) (Avanti Polar Lipids, #880001), 1,2-dipalmitoyl- sn -glycero-3-phosphate (di-C 16:0 PA; Avanti Polar Lipids, #830855), 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) (Anatrace, #D514), and 1,2-diheptanoyl- sn -glycero-3-phosphocholine (DHPC) (Anatrace, #D607).

    Techniques: Titration, Labeling, Mutagenesis

    Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

    Journal: Frontiers in Pharmacology

    Article Title: Immune Checkpoint Inhibitors Regulate K + Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

    doi: 10.3389/fphar.2021.742862

    Figure Lengend Snippet: Differential time-dependent involvement of PI3K and calmodulin on PD-L1 mediated inhibition of KCa3.1 channels. (A,B) Representative recordings of KCa3.1 channels in activated CD8 + PBTs from HDs showing the effect of the PI3K inhibitor LY294002 (10 µM) +/− phosphatidylinositol-3 phosphatase (PI3P) (100 nM) (A) and PD-L1 (PD-L1-Fc, 10 μg/ml) +/− PI3P (100 nM) (B). (C) Summary of the pharmacological modulation of KCa3.1 channels byLY294002 and PI3P in the absence and presence of plate bound PD-L1 in activated CD8 + PBTs of HDs. Cells were activated using anti-CD3/CD28 antibodies for 72 h. Cells were perfused with LY294002 for 15 min followed by patch clamp recordings with and without PI3P, delivered intracellularly via patch pipette ( n = eight to nine cells per group from 3 HDs). All KCa3.1 conductance (G) values are normalized to the average G of the control group (drug-free). (D) KCa3.1 G measured in absence or presence of PD-L1 in activated CD8 + PBTs of HDs. Cells were treated with plate-bound PD-L1 (PD-L1-Fc, 10 μg/ml) and activated using anti-CD3/CD28 antibodies for 120 h PI3P was delivered intracellularly via the patch pipette during the electrophysiological experiments (drug-free control). Cells were held at −70 mV, n = four to five cells per group from one HD. The values in panel (C,D) are represented as box and whiskers plot. The lower and upper bound of the box represent 25 th and 75 th percentiles respectively. Median values are shown as horizontal line. The lower and upper error bars represents 10 th and 90 th percentile respectively. (E) Percentage change in mean fluorescence intensity (MFI) of ion channels (Kv1.3, KCa3.1, Orai1, Stim1) and Calmodulin (CaM) measured using flow cytometry. Each dot represents an individual HD and the horizontal black line represents the mean value. Data in panel (C) were analyzed by One Way ANOVA ( p < 0.001) followed by Holm-Sidak’s post hoc analysis. Data in (D) were analyzed by One way ANOVA followed by Holm-Sidak’s post hoc analysis.

    Article Snippet: Phosphatidylinositol 3-phosphate diC16 (PI3P) was purchased from Echelon Biosciences.

    Techniques: Inhibition, Patch Clamp, Transferring, Fluorescence, Flow Cytometry

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean equatorial GFP signal intensity in A–D ). (p=<0.0001; n = 20) ( F ) MCBs with PC/PS and PC/PS/CH lipid composition (0 mol% PI(3)P) incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Graph presents mean equatorial GFP signal intensity (p=0.005; n = 25). For both E ) and F ) GFP signal intensity is normalized to DiD signal intensity, however the same pattern can be seen in the raw intensity values.

    Journal: eLife

    Article Title: A non-linear system patterns Rab5 GTPase on the membrane

    doi: 10.7554/eLife.54434

    Figure Lengend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean equatorial GFP signal intensity in A–D ). (p=<0.0001; n = 20) ( F ) MCBs with PC/PS and PC/PS/CH lipid composition (0 mol% PI(3)P) incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Graph presents mean equatorial GFP signal intensity (p=0.005; n = 25). For both E ) and F ) GFP signal intensity is normalized to DiD signal intensity, however the same pattern can be seen in the raw intensity values.

    Article Snippet: Other , Phosphatidylinositol 3-phosphate diC16 (PI(3)P diC16) , Echelon , P-3016 , .

    Techniques: Incubation

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.

    Journal: eLife

    Article Title: A non-linear system patterns Rab5 GTPase on the membrane

    doi: 10.7554/eLife.54434

    Figure Lengend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.

    Article Snippet: Other , Phosphatidylinositol 3-phosphate diC16 (PI(3)P diC16) , Echelon , P-3016 , .

    Techniques: Incubation

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean GFP-Rab5 signal intensity outside of and within segmented domains in B ) and D ) (p=<0.0001) (See also ). ( F ) Mean GFP-Rab5 signal intensity outside of and within segmented domains on MCBs with PC/PS/CH/PlasmPE and PC/PS/CH/SM lipid composition containing 1 mol% PI(3)P (p=<0.0046) (See also ).

    Journal: eLife

    Article Title: A non-linear system patterns Rab5 GTPase on the membrane

    doi: 10.7554/eLife.54434

    Figure Lengend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the GFP channel ( right ). Scale Bar = 10 µm. ( E ) Mean GFP-Rab5 signal intensity outside of and within segmented domains in B ) and D ) (p=<0.0001) (See also ). ( F ) Mean GFP-Rab5 signal intensity outside of and within segmented domains on MCBs with PC/PS/CH/PlasmPE and PC/PS/CH/SM lipid composition containing 1 mol% PI(3)P (p=<0.0046) (See also ).

    Article Snippet: Other , Phosphatidylinositol 3-phosphate diC16 (PI(3)P diC16) , Echelon , P-3016 , .

    Techniques: Incubation

    MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.

    Journal: eLife

    Article Title: A non-linear system patterns Rab5 GTPase on the membrane

    doi: 10.7554/eLife.54434

    Figure Lengend Snippet: MCBs with PC/PS and EE lipid composition containing 1 mol% PI(3)P ( A ) and B ) respectively) and MCBs with PC/PS and EE lipid composition containing 0 mol% PI(3)P ( C ) and D ) respectively) were incubated with 10 nM GFP-Rab5/GDI, 1 μM GDI, 100 nM Rabex5/Rabaptin5-RFP and 1 mM GTP for 15 min at 23 °C. Beads are presented as equatorial slices in GFP and DiD channels ( left ) and Mollweide projection of the DiD channel ( right ). Scale Bar = 10 µm.

    Article Snippet: Other , Phosphatidylinositol 3-phosphate diC16 (PI(3)P diC16) , Echelon , P-3016 , .

    Techniques: Incubation

    Journal: eLife

    Article Title: A non-linear system patterns Rab5 GTPase on the membrane

    doi: 10.7554/eLife.54434

    Figure Lengend Snippet:

    Article Snippet: Other , Phosphatidylinositol 3-phosphate diC16 (PI(3)P diC16) , Echelon , P-3016 , .

    Techniques: Recombinant, Expressing, Plasmid Preparation, Ligation, Transfection, Construct, Microscopy, BIA-KA