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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    Average 93 stars, based on 1 article reviews
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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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    pi 3 p p 3016  (Echelon Biosciences)


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    Echelon Biosciences pi 3 p p 3016
    Pi 3 P P 3016, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phosphatidylinositol 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

    dic16 pi3p  (Echelon Biosciences)


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    Echelon Biosciences dic16 pi3p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
    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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    dic16 pi3p - by Bioz Stars, 2024-04
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    1) Product Images from "PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase"

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    Journal: bioRxiv

    doi: 10.1101/2022.06.18.496676

    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
    Figure Legend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Expressing, Immunoprecipitation, Western Blot, Kinase Assay, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.
    Figure Legend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Techniques Used: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .
    Figure Legend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Techniques Used: Autoradiography, In Vitro, Mass Assay, Immunoprecipitation

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.
    Figure Legend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Techniques Used:

    (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.
    Figure Legend Snippet: (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Techniques Used:

    dic16 pi3p echelon p  (Echelon Biosciences)


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    Echelon Biosciences dic16 pi3p echelon p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
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    1) Product Images from "PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase"

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    Journal: bioRxiv

    doi: 10.1101/2022.06.18.496676

    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
    Figure Legend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Techniques Used: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Expressing, Immunoprecipitation, Western Blot, Kinase Assay, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.
    Figure Legend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Techniques Used: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .
    Figure Legend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Techniques Used: Autoradiography, In Vitro, Mass Assay, Immunoprecipitation

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.
    Figure Legend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Techniques Used:

    (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.
    Figure Legend Snippet: (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Techniques Used:

    pi 3 p  (Echelon Biosciences)


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    Echelon Biosciences pi 3 p
    Pi 3 P, 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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    di c16 ptdins 3 p  (Echelon Biosciences)


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    Echelon Biosciences di c16 ptdins 3 p
    ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide  with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.
    Di C16 Ptdins 3 P, 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 "De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex"

    Article Title: De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex

    Journal: Science Advances

    doi: 10.1126/sciadv.abg4007

    ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide  with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.
    Figure Legend Snippet: ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Binding Assay

    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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    pi 3 pdic16  (Echelon Biosciences)


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    Echelon Biosciences pi 3 pdic16
    Phosphoinositide-binding domains
    Pi 3 Pdic16, 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 "Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro"

    Article Title: Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro

    Journal: STAR Protocols

    doi: 10.1016/j.xpro.2021.100926

    Phosphoinositide-binding domains
    Figure Legend Snippet: Phosphoinositide-binding domains

    Techniques Used: Binding Assay

    ING2 binds PI(5)P Exogenous PI, PI(3)P, PI(3,5)P 2 or PI(5)P (100 μM) were spotted on a membrane. The membrane was incubated with recombinant GST-ING2 (10 nM). Anti-GST antibody was used to visualize GST-ING2 binding to the lipids on the membrane. Where necessary, consider comparing signals from GST-tagged lipid probe to GST alone.
    Figure Legend Snippet: ING2 binds PI(5)P Exogenous PI, PI(3)P, PI(3,5)P 2 or PI(5)P (100 μM) were spotted on a membrane. The membrane was incubated with recombinant GST-ING2 (10 nM). Anti-GST antibody was used to visualize GST-ING2 binding to the lipids on the membrane. Where necessary, consider comparing signals from GST-tagged lipid probe to GST alone.

    Techniques Used: Incubation, Recombinant, Binding Assay


    Figure Legend Snippet:

    Techniques Used: Recombinant, Protease Inhibitor, Staining, In Vitro, Magnetic Beads, Bicinchoninic Acid Protein Assay, Software

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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 dic16 pi3p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
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    Echelon Biosciences dic16 pi3p echelon p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
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    Echelon Biosciences pi 3 p
    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, <t>PI3P:</t> <t>Phosphatidylinositol</t> <t>3-phosphate,</t> <t>PI(3,5)P</t> 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.
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    Echelon Biosciences di c16 ptdins 3 p
    ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide  with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.
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    Echelon Biosciences p 3016
    ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide  with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.
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    Echelon Biosciences pi 3 pdic16
    Phosphoinositide-binding domains
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    Image Search Results


    ( 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

    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 µM Phosphatidylserine (PS) (Sigma #P5660) and dried in a centrifugal vacuum concentrator.

    Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Expressing, Immunoprecipitation, Western Blot, Kinase Assay, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 µM Phosphatidylserine (PS) (Sigma #P5660) and dried in a centrifugal vacuum concentrator.

    Techniques: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 µM Phosphatidylserine (PS) (Sigma #P5660) and dried in a centrifugal vacuum concentrator.

    Techniques: Autoradiography, In Vitro, Mass Assay, Immunoprecipitation

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 µM Phosphatidylserine (PS) (Sigma #P5660) and dried in a centrifugal vacuum concentrator.

    Techniques:

    (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Article Snippet: diC16-PI3P (Echelon) was mixed with 20 µM Phosphatidylserine (PS) (Sigma #P5660) and dried in a centrifugal vacuum concentrator.

    Techniques:

    (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) Schematic illustrating the putative enzymatic routes by which PI5Pcan be synthesised in Drosophila . The activities of enzymes labelled in blue are known in mammalian cells, the activity of enzymes labelled in red followed by “?” are still unknown in Drosophila , the activity of enzymes labelled in green are known in Drosophila . The activity of PI5P to PI(4,5)P 2 is boxed and is linked to cell size regulation in Drosophila . PI: Phosphatidylinositol, PI3P: Phosphatidylinositol 3-phosphate, PI(3,5)P 2 : Phosphatidylinositol 3,5 bisphosphate, PI5P: Phosphatidylinositol 5-phosphate, PI(4,5)P 2 : Phosphatidylinositol 4,5 bisphosphate. (B) Schematic illustrating the LC-MS/MS based in vitro 5-kinaseactivity assay using S2R+ cells over-expressing dFab1 enzyme to convert synthetic PI or PI3P to PI5P and PI(3,5)P 2 , respectively. (C) (i) Immunoprecipitated protein levels were analysed by Western blotting with an anti-mCherry antibody. Control (IgG) was prepared without anti-mCherry. Input lane shows the correct size of dFab1 protein ∼230 kDa. UTC: Untransfected control. (ii) In vitro kinase assay on synthetic PI and PI3P. Graph representing the Kinase activity (%) as the normalised response ratio of PI 5-kinase activity on PI” to PI3P 5-kinase activity on PI3P” upon enzymatic activity of immunoprecipitated mCherry::dFab1 on the respective substrates. Response ratio of PI 5-kinase activity on PI is obtained from area under the curve (AUC) of 17:0 14:1 PI5P (Product)/17:0 14:1 PI (Substrate), Response ratio of PI3P 5-kinaseactivity on PI3P isobtained from area under the curve (AUC) of 17:0 20:4 PI(3,5)P 2 (Product)/17:0 20:4 PI3P (Substrate) and is represented as mean ± S.E.M. on addition of either negative control (no beads), Control (mCherry beads) or dFab1 (mCherry::dFab1 beads). Number of immunoprecipitated samples= 2. (D) Schematic illustrating the LC-MS/MS based in vitro PI(3,5)P 2 3-phosphataseactivity assay using dsRNA treated S2R+ cells as enzyme source to convert synthetic PI(3,5)P 2 [d5-PI(3,5)P 2 to d5- 18 O-PIP 2 ] using a two-step reaction scheme. (E) In vitro phosphatase assay on synthetic PI(3,5)P 2 . Graph representing the 3-Phosphatase activity (%) as the percent formation of d5- 18 O-PIP 2 formed from starting d5-PI(3,5)P 2 as mean ± S.E.M. on addition of either control (GFPdsRNA) or Mtm, CG3632, CG3530 dsRNA treated S2R+ cell lysates. One way ANOVA with a post hoc Tukey’s test shows p value = 0.003 between GFP and Mtm ds RNA treated lysates, shows p value = 0.63 between GFP and CG3632 ds RNA treated lysate and shows p value= 0.11 between GFP and CG3530 dsRNA treated lysates.

    Article Snippet: diC16-PI3P – Echelon P-3016 ; diC16-PI4P – Echelon P-4016; Avanti 850172 | rac-16:0 PI(3,5)P 2 -d5 (Custom synthesised); 17: 0 20: 4 PI3P - Avanti LM-1900; 17: 0 20: 4; PI(4,5)P 2 - Avanti LM-1904.

    Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, In Vitro, Expressing, Immunoprecipitation, Western Blot, Kinase Assay, Negative Control, Phosphatase Assay

    (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) In vitro phosphatase assay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed as mean ± S.E.M. on addition of either control (da/+) or da>Mtm WT GFP lysates. Lysate samples= 3, where each sample was made from fivethird instar wandering larvae. Strudent’s Unpaired t-test with Welch correction showed p value= 0.007. (B) Extracted ion chromatogram (XIC) of deacylated PI3P or GroPI3P (Glycerophosphoinositol 3-phosphate) peak at Rt = 7.37 min, separated from deacylated PI4P or GroPI4P (Glycerophosphoinositol 4-phosphate) peak at Rt = 9.12 min obtained from injecting wild type larval lipid extract (details of sample preparation is discussed in methods). (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+; dPIP4K 29 (green) and da> Mtm WT GFP, dPIP4K 29 (majenta). Biological samples = 3, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of da/+ (green) andda> Mtm WT GFP, (majenta). Biological samples= 3, where each sample was made from three third instar wandering larvae.. Student’s unpaired t-test with Welch correction showed p value = 0.13.

    Article Snippet: diC16-PI3P – Echelon P-3016 ; diC16-PI4P – Echelon P-4016; Avanti 850172 | rac-16:0 PI(3,5)P 2 -d5 (Custom synthesised); 17: 0 20: 4 PI3P - Avanti LM-1900; 17: 0 20: 4; PI(4,5)P 2 - Avanti LM-1904.

    Techniques: In Vitro, Phosphatase Assay, Sample Prep

    (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of wild type (green) and dPIP4K 29 (majenta). Biological samples = 5, where each sample was made from five third instar wandering larvae. Unpaired t-test with Welch correction showed p value= 0.008. (B) Autoradiograph of TLC ran with lipid samples from in vitro PI3Pmassassay using wild type(WT) and dPIP4K 29 lipid samples. The first two lanes from the left are obtained from mass assay reactions using synthetic PI3P standard without or with addition of dFab1 enzyme, respectively. The origin spot and PI(3,5)P 2 spots are marked. (C) The graph represents normalised PI3P levels. Briefly, the spot marked asPI(3,5)P 2 on TLC in (B) is obtained by converting PI3P in the samples using immunoprecipitated dFab1 in presence of ϒ 32 P-ATP are quantified using image analysis and then normalised to organic phosphate value (indicated in blue embedded text under TLC) to obtain normalised PI3P levels. Biological samples = 3, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.036. (D) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.008. (E) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organicphosphatevalueof total lipid extractsasmean ± S.E.M. of Act5C/+; dPIP4K 29 (green) or Act5C> dPIP4K D271A , dPIP4K 29 (majenta). Biological samples = 6, where each sample was made from three third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.818. (F) In vitro phosphataseassay on synthetic PI3P. Graph representing the response ratio of 17:0 20:4 PI (Product)/17:0 20:4 PI3P (Substrate) formed asmean ± S.E.M. on addition of either wildtype (WT) or dPIP4K 29 lysatesfor either a 5 min or a 15 min reaction. Lysate samples= 3, where each sample was made from five third instar wandering larvae. Multiple unpaired t-test showed p value = 0.26 for 5 min time point and p value= 0.052. (G) qPCR measurements for mRNA levels of PI3K59F and PI3K68D from either Wild type (green) or dPIP4K 29 (majenta). Student’s unpaired t-test showed p value= 0.01 for PI3K59F and p value = 0.58 for PI3K68D . qPCR measurements for mRNA levels of Mtm, CG3632 and CG3530 from either Wild type (green) or dPIP4K 29 (majenta). Student’s unparied t-test showed p value = 0.03 for Mtm , p value= 0.23 for CG3632 and p value= 0.0006 for CG3530 .

    Article Snippet: diC16-PI3P – Echelon P-3016 ; diC16-PI4P – Echelon P-4016; Avanti 850172 | rac-16:0 PI(3,5)P 2 -d5 (Custom synthesised); 17: 0 20: 4 PI3P - Avanti LM-1900; 17: 0 20: 4; PI(4,5)P 2 - Avanti LM-1904.

    Techniques: Autoradiography, In Vitro, Mass Assay, Immunoprecipitation

    (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value<0.0001. (B) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4Pnormalised to organic phosphate value of total lipid extracts as mean ± S.E.M. of Act5C/+; dPIP4K 29 (green), or Act5C> dPIP4K WT GFP, dPIP4K 29 (majenta). Biological samples= 5, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.011. (C) (i) Representative confocal z-projections depicting a sub population of early endosomal compartment using 2xFYVE-mCherry in the salivary glands from wandering third instar larvaeof AB1> 2xFYVE-mCherry and AB1> 2xFYVE-mCherry ; dPIP4K 29 . Scalebar indicated at 20 μm. (ii) Graph representing 2xFYVE punctaemeasurement in the s alivary glands from wandering third instar larvae of AB1> 2xFYVEmCherry (N = 8, n=40) and AB1> 2xFYVEmCherry ; dPIP4K 29 (N =8, n = 40). Student’s unpaired t-test with Welch correction showed p value= 0.4057 (D) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 8), AB1>dPIP4K 2XFYVE ; dPIP4K 29 (n = 8). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value= 0.171. (E) (i) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the wandering third instar larvae of AB1>ATG8a-mCherry and AB1>ATG8a-mCherry; dPIP4K 29 . Scale bar indicated at 20 μm. (ii) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N = 10, n = 60) and AB1>ATG8a-mCherry; dPIP4K 29 (N = 10, n = 62). Student’s unpaired t-test with Welch correction showed p value<0.0001. (F) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvaeof AB1/+; dPIP4K 29 (n = 9), AB1>PI3K59F RNAi ; dPIP4K 29 (n = 9). Sample size is represented on individual bars. ired t-test with Welch correction showed p value<0.0001.

    Article Snippet: diC16-PI3P – Echelon P-3016 ; diC16-PI4P – Echelon P-4016; Avanti 850172 | rac-16:0 PI(3,5)P 2 -d5 (Custom synthesised); 17: 0 20: 4 PI3P - Avanti LM-1900; 17: 0 20: 4; PI(4,5)P 2 - Avanti LM-1904.

    Techniques:

    (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Journal: bioRxiv

    Article Title: PI3P dependent regulation of cell size by phosphatidylinositol 5-phosphate 4-kinase

    doi: 10.1101/2022.06.18.496676

    Figure Lengend Snippet: (A) qPCR measurements for mRNA levels of mtm, CG3632 and CG3530 from either Control ( daGal4 /+, in green) or da > Mtm RNAi, in majenta. Multiple t-test with post hoc Holm-Sidak’s test showed p value< 0.0001 between daGal4 /+ and da > Mtm RNAi for Mtm and p value= 0.35 between daGal4 /+ and da > Mtm RNAi for CG3632 , and p value = 0.04 between daGal4 /+ and da > Mtm RNAi for CG3530 . (B) (i) Representative confocal images of salivary glands from the genotypes a. AB1Gal4/+, b. AB1>Mtm RNAi. Cell body is marked majenta by BODIPY conjugated lipid dye, nucleus is marked by DAPI shown in green. Scale bar indicated at 50 μm. (ii) Graph representing average cell size measurement (in μm 3 ) asmean ± S.E.M. of salivary glands from wandering third instar larvae of AB1Gal4/+ (n = 7), AB1> Mtm RNAi (n = 7). Sample size is represented on individual bars. Student’s unpaired t-test with Welch correction showed p value = 0.0005. (C) Graph representing Normalised PI3P levels which is the peak area of GroPI3P/ peak area of GroPI4P normalised to organic phosphate value as mean ± S.E.M. of da /+ (green) and da> Mtm RNAi (majenta). Biological samples = 4, where each sample was made from five third instar wandering larvae. Student’s unpaired t-test with Welch correction showed p value= 0.07. (D) (i) Graph representing Atg8a punctae measurement in the salivary glands from wandering third instar larvaeof AB1>ATG8a-mCherry (N =7, n =40), AB1>ATG8a-mCherry; Mtm RNAi (N =7, n =40). Student’s unpaired t-test with Welch correction showed p value<0.0001. (ii) Representative confocal z-projections depicting autophagosomal levels using Atg8a-mCherry in the salivary glands from the genotypesa. AB1>ATG8a-mCherry, b. AB1>ATG8a-mCherry; Mtm RNAi. Scale bar indicated at 20 μm. (E) Graph representing average cell size measurement (in μm 3 ) as mean ± S.E.M. of salivary glands from wandering third instar larvae of AB1/+ (n = 11), AB1>Mtm RNAi (n = 8), AB1>Mtm RNAi, Atg8a RNAi (n = 12). Sample size is represented on individual bars. One way ANOVA with post hoc Tukey’s test showed p value < 0.0001 between AB1/+ and AB1>Mtm RNAi and p value = 0.0002 between AB1/+; dPIP4K 29 and AB1>Mtm RNAi, Atg8a RNAi.

    Article Snippet: diC16-PI3P – Echelon P-3016 ; diC16-PI4P – Echelon P-4016; Avanti 850172 | rac-16:0 PI(3,5)P 2 -d5 (Custom synthesised); 17: 0 20: 4 PI3P - Avanti LM-1900; 17: 0 20: 4; PI(4,5)P 2 - Avanti LM-1904.

    Techniques:

    ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide  with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.

    Journal: Science Advances

    Article Title: De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex

    doi: 10.1126/sciadv.abg4007

    Figure Lengend Snippet: ( A ) Interactions of retromer with TBC1D5, SNX3, SNX27, and Fam21 in the presence of either RT-D3 or RT-L4. GST-TBC1D5 TBC and GST-Fam21 R19-R21 were used as baits for retromer, while GST-tagged retromer (Vps29 subunit) was used as bait for SNX3 and SNX27. ( B ) ITC measurement of TBC1D5 TBC , ( C ) SNX3 (with DMT1-II 550–568 present), ( D ) SNX27 PDZ , and ( E ) GST-Fam21 R19-R21 with retromer in the presence or absence of RT-D3 or RT-L4. ( F ) Hela cell lysates were incubated with streptavidin agarose coated with biotinylated RT-D3 or RT-L4 and bound proteins subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting with antibodies against indicated proteins. ( G ) ITC measurement of PTHR 586–593 cargo peptide with SNX27 PDZ alone (yellow), retromer + RT-L4 (red), retromer + SNX27 PDZ (green), and retromer + SNX27 PDZ + RT-L4 (blue). RT-L4 allosterically enhances the affinity of retromer + SNX27 PDZ for cargo. The cargo peptide binds to SNX27. All ITC graphs represent the integrated and normalized data fit with a 1-to-1 binding ratio. The binding affinity ( K d ) is given as means of at least two independent experiments. ( H ) Liposome binding assay of retromer with membrane-associated SNX3-cargo complex in the presence of cyclic peptides. Multilamellar vesicles were composed of either control PC/PE lipids or Folch I lipids containing added PtdIns(3) P and N-terminal palmitoylated CI-MPR 2347–2376 peptide (schematic diagram on top). “S” and “P” indicate unbound supernatant and bound pellet, respectively. Control experiments are shown in fig. S7 (B and C). ( I ) Schematic summarizing the effects of RT-D3 and RT-L4 on retromer engagement with known regulatory, adaptor, and bacterial effector proteins.

    Article Snippet: Briefly, cargo-loaded Folch liposomes were made by mixing 25 μl of N-terminal palmitoylated CI-MPR 2347–2376 peptide (4 mg/ml), 50 μl of Folch fraction I (Sigma-Aldrich) (10 mg/ml), and 50 μl of di-C16 PtdIns(3) P (1 mg/ml) (Echelon Biosciences), each freshly prepared in chloroform, to a total volume of 500 μl of chloroform.

    Techniques: Incubation, Polyacrylamide Gel Electrophoresis, Western Blot, Binding Assay

    Phosphoinositide-binding domains

    Journal: STAR Protocols

    Article Title: Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro

    doi: 10.1016/j.xpro.2021.100926

    Figure Lengend Snippet: Phosphoinositide-binding domains

    Article Snippet: PIdiC16, PI(3)PdiC16, PI(3,5)P2diC16 , Echelon , #P-0016 #P-3016 #P-3516.

    Techniques: Binding Assay

    ING2 binds PI(5)P Exogenous PI, PI(3)P, PI(3,5)P 2 or PI(5)P (100 μM) were spotted on a membrane. The membrane was incubated with recombinant GST-ING2 (10 nM). Anti-GST antibody was used to visualize GST-ING2 binding to the lipids on the membrane. Where necessary, consider comparing signals from GST-tagged lipid probe to GST alone.

    Journal: STAR Protocols

    Article Title: Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro

    doi: 10.1016/j.xpro.2021.100926

    Figure Lengend Snippet: ING2 binds PI(5)P Exogenous PI, PI(3)P, PI(3,5)P 2 or PI(5)P (100 μM) were spotted on a membrane. The membrane was incubated with recombinant GST-ING2 (10 nM). Anti-GST antibody was used to visualize GST-ING2 binding to the lipids on the membrane. Where necessary, consider comparing signals from GST-tagged lipid probe to GST alone.

    Article Snippet: PIdiC16, PI(3)PdiC16, PI(3,5)P2diC16 , Echelon , #P-0016 #P-3016 #P-3516.

    Techniques: Incubation, Recombinant, Binding Assay

    Journal: STAR Protocols

    Article Title: Protocol for determining the regulation of lipid kinases and changes in phospholipids in vitro

    doi: 10.1016/j.xpro.2021.100926

    Figure Lengend Snippet:

    Article Snippet: PIdiC16, PI(3)PdiC16, PI(3,5)P2diC16 , Echelon , #P-0016 #P-3016 #P-3516.

    Techniques: Recombinant, Protease Inhibitor, Staining, In Vitro, Magnetic Beads, Bicinchoninic Acid Protein Assay, Software