pi 3 p dic16  (Echelon Biosciences)


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    Echelon Biosciences pi 3 p dic16
    CvpE binds <t>PI(3)P</t> and perturbs PIKfyve activity on lysosomes.
    Pi 3 P Dic16, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Coxiella burnetii effector CvpE maintains biogenesis of Coxiella -containing vacuoles by suppressing lysosome tubulation through binding PI(3)P and perturbing PIKfyve activity on lysosomes"

    Article Title: Coxiella burnetii effector CvpE maintains biogenesis of Coxiella -containing vacuoles by suppressing lysosome tubulation through binding PI(3)P and perturbing PIKfyve activity on lysosomes

    Journal: Virulence

    doi: 10.1080/21505594.2024.2350893

    CvpE binds PI(3)P and perturbs PIKfyve activity on lysosomes.
    Figure Legend Snippet: CvpE binds PI(3)P and perturbs PIKfyve activity on lysosomes.

    Techniques Used: Activity Assay

    mouse anti pi 3 p antibody  (Echelon Biosciences)


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    Echelon Biosciences mouse anti pi 3 p antibody
    ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or <t>PI(3)P</t> (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Mouse Anti Pi 3 P Antibody, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol"

    Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

    Journal: Science Advances

    doi: 10.1126/sciadv.adl6082

    ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or PI(3)P (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
    Figure Legend Snippet: ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or PI(3)P (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Techniques Used: Two Tailed Test, Knock-Out, Expressing, Incubation, Staining, Fluorescence, FACS, Transfection, Over Expression, Construct

    ( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.
    Figure Legend Snippet: ( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

    Techniques Used: Immunostaining, shRNA, Expressing, Incubation, Two Tailed Test, Immunoprecipitation, Mutagenesis, Transfection, Over Expression, Construct

    pi 3 p mass elisa kit  (Echelon Biosciences)


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    Echelon Biosciences pi 3 p mass elisa kit
    Pi 3 P Mass Elisa Kit, 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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    bodipy fl dic 6 pi 3 4 p 2  (Echelon Biosciences)


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    Echelon Biosciences bodipy fl dic 6 pi 3 4 p 2
    IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.
    Bodipy Fl Dic 6 Pi 3 4 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring"

    Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.4c00117

    IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.
    Figure Legend Snippet: IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.

    Techniques Used: Titration, Binding Assay, Comparison

    Fluorescence polarization study to assess IP6 as a competitive inhibitor. (A) Binding of tracer BODIPY-PI(3,4)P 2 to p47 phox -PX and associated fit for K d determination. (B) Displacement of BODIPY-PI(3,4)P 2 tracer from the PX domain upon addition of IP6 and associated fit. Error bars represent standard deviation of experimental replicates; those not visible are smaller than the data points.
    Figure Legend Snippet: Fluorescence polarization study to assess IP6 as a competitive inhibitor. (A) Binding of tracer BODIPY-PI(3,4)P 2 to p47 phox -PX and associated fit for K d determination. (B) Displacement of BODIPY-PI(3,4)P 2 tracer from the PX domain upon addition of IP6 and associated fit. Error bars represent standard deviation of experimental replicates; those not visible are smaller than the data points.

    Techniques Used: Fluorescence, Binding Assay, Standard Deviation

    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti pi 3 4 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 3 4 p 2
    Anti Pi 3 4 P 2, 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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    dic8 pi 3 p p 3008  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 p p 3008
    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Dic8 Pi 3 P P 3008, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer"

    Article Title: Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

    Journal: The EMBO Journal

    doi: 10.1038/s44318-024-00085-6

    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Figure Legend Snippet: ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Techniques Used: Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy

    dic8 pi 3 4 p2 p 3408  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 4 p2 p 3408
    Dic8 Pi 3 4 P2 P 3408, 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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    dic8 pi 3 5 p 2 p 3508  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 5 p 2 p 3508
    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Dic8 Pi 3 5 P 2 P 3508, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer"

    Article Title: Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

    Journal: The EMBO Journal

    doi: 10.1038/s44318-024-00085-6

    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Figure Legend Snippet: ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Techniques Used: Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy

    dic8 pi 3 p p 3008  (Echelon Biosciences)


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    Echelon Biosciences dic8 pi 3 p p 3008
    Dic8 Pi 3 P P 3008, 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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    Echelon Biosciences pi 3 p dic16
    CvpE binds <t>PI(3)P</t> and perturbs PIKfyve activity on lysosomes.
    Pi 3 P Dic16, 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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    ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or <t>PI(3)P</t> (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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    ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or <t>PI(3)P</t> (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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    Echelon Biosciences bodipy fl dic 6 pi 3 4 p 2
    IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.
    Bodipy Fl Dic 6 Pi 3 4 P 2, 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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    IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.
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    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Dic8 Pi 3 P P 3008, 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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    86
    Echelon Biosciences dic8 pi 3 4 p2 p 3408
    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Dic8 Pi 3 4 P2 P 3408, 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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    86
    Echelon Biosciences dic8 pi 3 5 p 2 p 3508
    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM <t>diC8</t> PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.
    Dic8 Pi 3 5 P 2 P 3508, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dic8 pi 3 5 p 2 p 3508/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dic8 pi 3 5 p 2 p 3508 - by Bioz Stars, 2024-05
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    Image Search Results


    CvpE binds PI(3)P and perturbs PIKfyve activity on lysosomes.

    Journal: Virulence

    Article Title: Coxiella burnetii effector CvpE maintains biogenesis of Coxiella -containing vacuoles by suppressing lysosome tubulation through binding PI(3)P and perturbing PIKfyve activity on lysosomes

    doi: 10.1080/21505594.2024.2350893

    Figure Lengend Snippet: CvpE binds PI(3)P and perturbs PIKfyve activity on lysosomes.

    Article Snippet: The following chemicals were used in this study: chloramphenicol (Millipore 220,551), Vacuolin-1 (Selleck, S6912), YM201636 (MC E, HY-13228), ML-SA5 (MCE, HY-152182), and PI(3)P diC16 (Echelon Biosciences, p -3016).All recombinant His or GST tagged fusion proteins involved in this article were purchased from CUSABIO (Wuhan, China).

    Techniques: Activity Assay

    ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or PI(3)P (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Journal: Science Advances

    Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

    doi: 10.1126/sciadv.adl6082

    Figure Lengend Snippet: ( A ) LC3 puncta in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 3, two-tailed paired Student’s t test. Data are means ± SEM. ( B ) HTTQ74-EGFP aggregates in control or ATG16 knockout (KO) HeLa cells, treated with p47 siRNA or control. The number of HTTQ74-EGFP aggregates represents the number of cells containing visible aggregates in cells positive for EGFP signal; n = 4, two-tailed paired Student’s t test. Data are means ± SEM. ( C ) LC3-II levels in control and p47 siRNA–treated HeLa cells, treated with 400 nM of BafA1 for 4 hours; n = 4, one-sample t test. Data are means ± SEM. ( D ) Protein levels in HeLa cells treated with control or p47 siRNA. ( E and F ) Control, p47 KO cells, or p47 KO cells expressing p47 wild-type (WT) were incubated in Earle’s balanced salt solution (EBSS) starvation media for 2 hours and stained for WIPI2 (E) or PI(3)P (F); n = 3 to 6, one-way ANOVA ( P = 0.0005) with post hoc Tukey test for (E), one-way ANOVA ( P = 0.0245) with post hoc Tuckey test for (F), data are means ± SEM. ( G ) HeLa cells expressing SRAI-LC3B were treated with control, p47, or ATG7 siRNA for 72 hours before treatment with 20 μM SMER28 for 24 hours, followed by fluorescence-activated cell sorting (FACS) analysis; n = 9, one-way ANOVA ( P = 0.0005) with post hoc Tukey test, data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs. ns, not significant; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

    Article Snippet: Mouse anti-PI(3)P antibody (1:300; 1 hour at room temperature) (Echelon Biosciences catalog no. Z-P003, RRID:AB_427221) and secondary antibody (1:400; 30 min at room temperature) (goat anti-mouse Alexa Fluor 555) were applied, followed by post-fixation in 2% paraformaldehyde, washing and mounting on microscope slides with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific).

    Techniques: Two Tailed Test, Knock-Out, Expressing, Incubation, Staining, Fluorescence, FACS, Transfection, Over Expression, Construct

    ( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

    Journal: Science Advances

    Article Title: p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol

    doi: 10.1126/sciadv.adl6082

    Figure Lengend Snippet: ( A ) Control, p37 KO, and p37 KO HeLa cells reconstituted with p37-FLAG were treated with 400 nM BafA1 for 4 hours, followed by immunostaining for LC3 and FLAG; n = 3, one-way ANOVA ( P = 0.004) with post hoc Tukey test. ( B ) iNeurons treated with lentiviral-delivered p37 shRNA (#81 or #83) for 4 days were treated with 400 nM BafA1 for 6 hours; n = 4, one-sample t test. ( C ) Control, p37 KO, and p37 KO with p37-FLAG–expressing HeLa cells were incubated in EBSS for 2 hours, followed by immunostaining for PI(3)P; n = 3 to 4, one-sample t test and two-tailed unpaired Student’s t test. ( D and E ) Endogenous ATG14L was immunoprecipitated from control and p37 KO HeLa cells (D) or control and p37-Clover–overexpressing HeLa cells (E); n = 4 to 5; one-sample t test. ( F to H ) Control or p37-FLAG–overexpressing cells were incubated in EBSS for 2 hours (F and G) and treated with 5 μM CB-5083 for 3 hours (G) or 400 nM BafA1 for 4 hours (H) where indicated, followed by immunostaining for PI(3)P (F), WIPI2 (G), or LC3 (H); n = 3, two-tailed unpaired Student’s t test. ( I ) HeLa cells expressing WT or SHP mutant p37-FLAG for 24 hours, followed by treatment with 400 nM BafA1 for 4 hours; n = 3 to 4, one-sample t test. ( J ) Control or Beclin-1 KO HeLa cells expressing p37-FLAG were treated with 400 nM BafA1 for 4 hours; n = 5, one-sample t test. Data are means ± SEM. Scale bars, 10 μm. In cDNA transfection experiments, matched empty vectors were used as controls for overexpression constructs, and in all knockdown experiments, we used nontargeting control siRNAs.

    Article Snippet: Mouse anti-PI(3)P antibody (1:300; 1 hour at room temperature) (Echelon Biosciences catalog no. Z-P003, RRID:AB_427221) and secondary antibody (1:400; 30 min at room temperature) (goat anti-mouse Alexa Fluor 555) were applied, followed by post-fixation in 2% paraformaldehyde, washing and mounting on microscope slides with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific).

    Techniques: Immunostaining, shRNA, Expressing, Incubation, Two Tailed Test, Immunoprecipitation, Mutagenesis, Transfection, Over Expression, Construct

    IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.

    Journal: Biochemistry

    Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

    doi: 10.1021/acs.biochem.4c00117

    Figure Lengend Snippet: IP6 binds to the PX domain of p47 phox as observed by NMR. (A) 1 H– 15 N-HSQC overlay of 1:0 and 1:2 molar ratios of p47 phox -PX:IP6. Insets depict a titration series including 1:0.25, 1:0.5, 1:2, and 1:4 molar ratios of PX domain:IP6. High-affinity binding is implicated through line-broadening of resonances. (B) Mapping of CSPs upon addition of 2× IP6 to the PX domain (PDB: 1GD5 ). Residues depicting 1 and 2 standard deviations of a 20% trimmed mean are in pink and purple, respectively. Residues with unobservable or line-broadened peaks are depicted in black. (C) Comparison of CSPs upon 2× C 4 –PI(3,4)P 2 (purple) or IP6 (orange). Residues with unobservable or line-broadened peaks are shown as red circles offset from the x -axis.

    Article Snippet: All fluorescence polarization (FP) experiments were performed using BODIPY-FL diC 6 –PI(3,4)P 2 [Echelon Biosciences (Salt Lake City, UT)] as the reporting tracer.

    Techniques: Titration, Binding Assay, Comparison

    Fluorescence polarization study to assess IP6 as a competitive inhibitor. (A) Binding of tracer BODIPY-PI(3,4)P 2 to p47 phox -PX and associated fit for K d determination. (B) Displacement of BODIPY-PI(3,4)P 2 tracer from the PX domain upon addition of IP6 and associated fit. Error bars represent standard deviation of experimental replicates; those not visible are smaller than the data points.

    Journal: Biochemistry

    Article Title: Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47 phox Membrane Anchoring

    doi: 10.1021/acs.biochem.4c00117

    Figure Lengend Snippet: Fluorescence polarization study to assess IP6 as a competitive inhibitor. (A) Binding of tracer BODIPY-PI(3,4)P 2 to p47 phox -PX and associated fit for K d determination. (B) Displacement of BODIPY-PI(3,4)P 2 tracer from the PX domain upon addition of IP6 and associated fit. Error bars represent standard deviation of experimental replicates; those not visible are smaller than the data points.

    Article Snippet: All fluorescence polarization (FP) experiments were performed using BODIPY-FL diC 6 –PI(3,4)P 2 [Echelon Biosciences (Salt Lake City, UT)] as the reporting tracer.

    Techniques: Fluorescence, Binding Assay, Standard Deviation

    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Journal: The EMBO Journal

    Article Title: Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

    doi: 10.1038/s44318-024-00085-6

    Figure Lengend Snippet: ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Article Snippet: Synthetic lipids diC8 PI (P-0008), diC8 PI(3)P (P-3008), diC8 PI(4)P (P-4008), diC8 PI(5)P (P-5008), diC8 PI(3,4)P 2 (P-3408), diC8 PI(3,5)P 2 (P-3508), diC8 PI(4,5)P 2 (P-4508), diC8 PI(3,4,5)P 3 (P-3908), diC16 PI(4,5)P 2 (P-4516), and diC16 PI(3,4,5)P 3 (P-3916) were purchased from Echelon Bioscience.

    Techniques: Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy

    ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Journal: The EMBO Journal

    Article Title: Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer

    doi: 10.1038/s44318-024-00085-6

    Figure Lengend Snippet: ( A ) The intensity of immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( B , C ) 0.05 μM His 6 -tagged PIPKIα ( B ) and IPMK ( C ) were incubated with 0.2 μM diC8 PI(4)P or diC8 PI(4,5)P 2 , respectively, in the absence or presence of various concentrations of GST-YAP (0.001, 0.01, 0.1, 1.0, and 10.0 μM). The activities of the kinases were measured using the ADP-Glo assay (Promega). The graphs show the mean±s.d. of n = 3 independent experiments. YAP did not alter the activity of either kinase. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( D ) Summary of GST alone or GST-YAP bindings to PI, PI(4,5)P 2 , or PI(3,4,5)P 3 measured by MST. Raw data are available in Appendix Fig. S . ( E ) A schematic representation of the molecular structure Ac 3 2API and how it can become metabolically incorporated into phosphoinositides after removal of acetyl groups by esterase to produce azido- myo -inositol probe 2API. ( F ) The intensity of the immunoblots of Fig. was quantified using ImageJ and the graph shows the mean ± s.d. of n = 3 independent experiments. * P < 0.05; ** P < 0.01, and n.s.; not significant in Student’s t test. ( G ) Starved MDA-MB-231 cells were fed with Ac 3 2API for 24 h in the presence of 10% dialyzed serum. Cells were lysed and azide-tagged molecules were conjugated to biotin–alkyne through a click reaction. Endogenous c-Myc was immunoprecipitated and the associated complexes were analyzed by immunoblotting. Representative immunoblot images of n = 2 independent experiments are shown. ( H ) The clicked lysates were analyzed by anti-PI(4,5)P 2 or PI(3,4,5)P 3 antibodies. Biotinylated 2API was resolved by streptavidin. Many immunoblot bands overlapped with streptavidin signals. Representative immunoblot images of n = 3 independent experiments are shown. ( I , J ) Starved MDA-MB-231 cells were stimulated with 10% serum for 1 h. The images are z-stacks of PI(4,5)P 2 -YAP PLA ( E ) and PI(3,4,5)P 3 -YAP PLA ( F ) taken using a confocal microscope with each frame differing by 0.2 μm. DAPI was used to stain the nucleus. Representative images of n = 3 independent experiments are shown. Scale bar, 10 μm. ( K , L ) MDA-MB-231 cells grown in 10% serum were stimulated with 5 μM LPA for 90 min. Cells were fixed and the association of YAP with PI(4,5)P 2 ( G ) or PI(3,4,5)P 3 ( H ) was visualized by PLA. The images were obtained by widefield epifluorescence microscopy. The number of PLA puncta was counted from at least 10 cells and the graph shows the mean±s.d. of n = 3 independent experiments. DAPI staining was used to distinguish the nucleus from the cytoplasm. Treating the cells with LPA significantly increased the number of nuclear puncta. Scale bar, 10 μm. * P < 0.05; ** P < 0.01, and n.s; not significant in Student’s t test.

    Article Snippet: Synthetic lipids diC8 PI (P-0008), diC8 PI(3)P (P-3008), diC8 PI(4)P (P-4008), diC8 PI(5)P (P-5008), diC8 PI(3,4)P 2 (P-3408), diC8 PI(3,5)P 2 (P-3508), diC8 PI(4,5)P 2 (P-4508), diC8 PI(3,4,5)P 3 (P-3908), diC16 PI(4,5)P 2 (P-4516), and diC16 PI(3,4,5)P 3 (P-3916) were purchased from Echelon Bioscience.

    Techniques: Western Blot, Incubation, Glo Assay, Activity Assay, Metabolic Labelling, Immunoprecipitation, Microscopy, Staining, Epifluorescence Microscopy