anti pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 3 5 p 2
    Anti Pi 3 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1 article reviews
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    anti pi 3 5 p 2 - by Bioz Stars, 2024-06
    92/100 stars

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    mouse anti pi 3 5 p 2 antibody  (Echelon Biosciences)


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    Echelon Biosciences mouse anti pi 3 5 p 2 antibody
    (A) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. PI(3,5)P 2 (red) was detected by immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of the relative intensity of PI(3,5)P 2 from (A) (n = 20). (C) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. PIKfyve, MTM1, and PRV gE were detected by immunoblotting. (D) PK-15 cells were mock-infected or infected with PRV HN1201 (MOI = 0.1) and treated with YM-201636 (10 μM) for 24 hours. PI(3,5)P 2 in the lysosomal fraction was detected by lipid dot-blot analysis. The fraction was analyzed by immunoblotting with an antibody against NPC1 (lysosome marker). (E) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. The colocalization of PI(3,5)P 2 (red) with LAMP1 (lysosomal marker, blue) was detected by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of colocalization of PI(3,5)P 2 with LAMP1 from (E) (n = 40). (G) PK-15 cells were treated with vehicle, YM-201636 (10 μM), or ML-SI1 (10 μM) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (H) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (I) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were incubated with PRV HN1201 (MOI = 0.1), vehicle, or poly-lysine (50 μg/mL) as indicated at 4°C for 2 hours, and then temperature-shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (J) Schematic representation of the rapamycin-inducible heterodimerization system used to recruit MTM1 to the lysosomal membrane. Rab7 is a lysosome-specific Rab protein. MTM1 is a PI 3 phosphatase that can convert PI(3,5)P 2 and PI(3)P into PI(5)P and PI, respectively. (K) HeLa cells were cotransfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. The subcellular localization of mCherry-FRB-MTM1 and BFP-FKBP-Rab7 was observed by fluorescence microscopy. Scale bar: 10 μm. (L) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. Intracellular Ca 2+ levels were detected using Fluo-3 staining (5 μM). Scale bar: 10 μm. (M) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes and infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (N) Mcoln1 +/+ and Mcoln1 -/- MEF cells were transfected with TRPML1-WT or TRPML1-7Q for 24 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). Data are expressed as the mean ± SD of 3 independent experiments. P -values were determined by Student’s t -test. P < 0.05 was considered statistically significant.
    Mouse Anti Pi 3 5 P 2 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
    https://www.bioz.com/result/mouse anti pi 3 5 p 2 antibody/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
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    86/100 stars

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    1) Product Images from "Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry"

    Article Title: Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry

    Journal: PLOS Pathogens

    doi: 10.1371/journal.ppat.1011956

    (A) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. PI(3,5)P 2 (red) was detected by immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of the relative intensity of PI(3,5)P 2 from (A) (n = 20). (C) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. PIKfyve, MTM1, and PRV gE were detected by immunoblotting. (D) PK-15 cells were mock-infected or infected with PRV HN1201 (MOI = 0.1) and treated with YM-201636 (10 μM) for 24 hours. PI(3,5)P 2 in the lysosomal fraction was detected by lipid dot-blot analysis. The fraction was analyzed by immunoblotting with an antibody against NPC1 (lysosome marker). (E) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. The colocalization of PI(3,5)P 2 (red) with LAMP1 (lysosomal marker, blue) was detected by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of colocalization of PI(3,5)P 2 with LAMP1 from (E) (n = 40). (G) PK-15 cells were treated with vehicle, YM-201636 (10 μM), or ML-SI1 (10 μM) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (H) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (I) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were incubated with PRV HN1201 (MOI = 0.1), vehicle, or poly-lysine (50 μg/mL) as indicated at 4°C for 2 hours, and then temperature-shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (J) Schematic representation of the rapamycin-inducible heterodimerization system used to recruit MTM1 to the lysosomal membrane. Rab7 is a lysosome-specific Rab protein. MTM1 is a PI 3 phosphatase that can convert PI(3,5)P 2 and PI(3)P into PI(5)P and PI, respectively. (K) HeLa cells were cotransfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. The subcellular localization of mCherry-FRB-MTM1 and BFP-FKBP-Rab7 was observed by fluorescence microscopy. Scale bar: 10 μm. (L) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. Intracellular Ca 2+ levels were detected using Fluo-3 staining (5 μM). Scale bar: 10 μm. (M) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes and infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (N) Mcoln1 +/+ and Mcoln1 -/- MEF cells were transfected with TRPML1-WT or TRPML1-7Q for 24 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). Data are expressed as the mean ± SD of 3 independent experiments. P -values were determined by Student’s t -test. P < 0.05 was considered statistically significant.
    Figure Legend Snippet: (A) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. PI(3,5)P 2 (red) was detected by immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of the relative intensity of PI(3,5)P 2 from (A) (n = 20). (C) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. PIKfyve, MTM1, and PRV gE were detected by immunoblotting. (D) PK-15 cells were mock-infected or infected with PRV HN1201 (MOI = 0.1) and treated with YM-201636 (10 μM) for 24 hours. PI(3,5)P 2 in the lysosomal fraction was detected by lipid dot-blot analysis. The fraction was analyzed by immunoblotting with an antibody against NPC1 (lysosome marker). (E) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. The colocalization of PI(3,5)P 2 (red) with LAMP1 (lysosomal marker, blue) was detected by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of colocalization of PI(3,5)P 2 with LAMP1 from (E) (n = 40). (G) PK-15 cells were treated with vehicle, YM-201636 (10 μM), or ML-SI1 (10 μM) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (H) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (I) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were incubated with PRV HN1201 (MOI = 0.1), vehicle, or poly-lysine (50 μg/mL) as indicated at 4°C for 2 hours, and then temperature-shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (J) Schematic representation of the rapamycin-inducible heterodimerization system used to recruit MTM1 to the lysosomal membrane. Rab7 is a lysosome-specific Rab protein. MTM1 is a PI 3 phosphatase that can convert PI(3,5)P 2 and PI(3)P into PI(5)P and PI, respectively. (K) HeLa cells were cotransfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. The subcellular localization of mCherry-FRB-MTM1 and BFP-FKBP-Rab7 was observed by fluorescence microscopy. Scale bar: 10 μm. (L) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. Intracellular Ca 2+ levels were detected using Fluo-3 staining (5 μM). Scale bar: 10 μm. (M) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes and infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (N) Mcoln1 +/+ and Mcoln1 -/- MEF cells were transfected with TRPML1-WT or TRPML1-7Q for 24 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). Data are expressed as the mean ± SD of 3 independent experiments. P -values were determined by Student’s t -test. P < 0.05 was considered statistically significant.

    Techniques Used: Infection, Immunofluorescence, Western Blot, Dot Blot, Marker, Staining, Incubation, Quantitative RT-PCR, Membrane, Fluorescence, Microscopy, Transfection

    anti pi 3 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 3 5 p 2
    Anti Pi 3 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 1 article reviews
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    anti pi 3 5 p 2 - by Bioz Stars, 2024-06
    92/100 stars

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    anti pi 4 5 p 2  (Echelon Biosciences)


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    Echelon Biosciences anti pi 4 5 p 2
    (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.
    Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pi 4 5 p 2/product/Echelon Biosciences
    Average 91 stars, based on 1 article reviews
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    1) Product Images from "Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia"

    Article Title: Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008317

    (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.
    Figure Legend Snippet: (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.

    Techniques Used: Binding Assay, Immunodetection

    (A-D) Light microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing Legionella -derived PIP-binding constructs. (A-B) Compared to low 2xFYVE-GFP-expressing cells from the same population, reduction of PI(3)P binding sites in cells highly expressing a regulated encystation-dependent epitope-tagged construct 2xFYVE-GFP (GFP) inhibits uptake of fluid-phase marker Dextran-R. Scale bars: 1 μm. (C-D) Expression levels of PI(4)P-binding epitope-tagged construct GFP-P4C expression (GFP) have no visible impact on Dextran-R signal at PVs of transfected cells. Scale bars: 1 μm. (E) Dextran-R uptake in non-transgenic wild-type cells as negative controls for construct-induced uptake phenotypes. Scale bars: 1 μm (F) Box-plot representing the distribution of cell width (in μm) across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) increase in median cell width with respect to non-transgenic cells is detected for 2xFYVE-GFP- but not GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (G) Box-plot representing the distribution of measured Dextran-R signal intensity across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal intensity, normalized to wild-type cells (100%), is detected for 2xFYVE-GFP- but not for GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (H) A statistically significant (p<0.5) linear correlation exists between Dextran-R signal ( x -axis, intensity_red channel [%]) and 2xFYVE-GFP expression ( y -axis, intensity_green channel [%]) measured across 100 cells. (I) The apparent linear correlation between GFP-P4C expression ( y -axis, intensity_green channel [%]) and Dextran-R signal ( x -axis, intensity_red channel [%]) is not statistically significant (p<0.5). (J) Wide-field microscopy-based immunofluorescence analysis of the impact of neomycin treatment on Dextran-R uptake to deplete PI(4,5)P 2 binding sites in non-transgenic wild-type cells. With respect to non-treated cells (WT; left panel), Dextran-R signal at PVs is visibly impacted in non-transgenic neomycin-treated cells (WT_Neo; right panel). Scale bars: 10 μm for full wide-field image, 1 μm for a single cell. (K) Box-plot representing the distribution of measured Dextran-R signal intensity across 100 wild-type cells, either untreated (WT) or treated with neomycin (WT_Neo). Neomycin treatment causes a statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal. Scale bars: wide-field: 10 μm; single cells: 1 μm. For all images, nuclei are labelled with DAPI (blue). Insets: DIC images.
    Figure Legend Snippet: (A-D) Light microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing Legionella -derived PIP-binding constructs. (A-B) Compared to low 2xFYVE-GFP-expressing cells from the same population, reduction of PI(3)P binding sites in cells highly expressing a regulated encystation-dependent epitope-tagged construct 2xFYVE-GFP (GFP) inhibits uptake of fluid-phase marker Dextran-R. Scale bars: 1 μm. (C-D) Expression levels of PI(4)P-binding epitope-tagged construct GFP-P4C expression (GFP) have no visible impact on Dextran-R signal at PVs of transfected cells. Scale bars: 1 μm. (E) Dextran-R uptake in non-transgenic wild-type cells as negative controls for construct-induced uptake phenotypes. Scale bars: 1 μm (F) Box-plot representing the distribution of cell width (in μm) across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) increase in median cell width with respect to non-transgenic cells is detected for 2xFYVE-GFP- but not GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (G) Box-plot representing the distribution of measured Dextran-R signal intensity across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal intensity, normalized to wild-type cells (100%), is detected for 2xFYVE-GFP- but not for GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (H) A statistically significant (p<0.5) linear correlation exists between Dextran-R signal ( x -axis, intensity_red channel [%]) and 2xFYVE-GFP expression ( y -axis, intensity_green channel [%]) measured across 100 cells. (I) The apparent linear correlation between GFP-P4C expression ( y -axis, intensity_green channel [%]) and Dextran-R signal ( x -axis, intensity_red channel [%]) is not statistically significant (p<0.5). (J) Wide-field microscopy-based immunofluorescence analysis of the impact of neomycin treatment on Dextran-R uptake to deplete PI(4,5)P 2 binding sites in non-transgenic wild-type cells. With respect to non-treated cells (WT; left panel), Dextran-R signal at PVs is visibly impacted in non-transgenic neomycin-treated cells (WT_Neo; right panel). Scale bars: 10 μm for full wide-field image, 1 μm for a single cell. (K) Box-plot representing the distribution of measured Dextran-R signal intensity across 100 wild-type cells, either untreated (WT) or treated with neomycin (WT_Neo). Neomycin treatment causes a statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal. Scale bars: wide-field: 10 μm; single cells: 1 μm. For all images, nuclei are labelled with DAPI (blue). Insets: DIC images.

    Techniques Used: Light Microscopy, Immunofluorescence, Transgenic Assay, Expressing, Derivative Assay, Binding Assay, Construct, Marker, Transfection, Microscopy

    anti pi 3 5 p 2 antibody  (Echelon Biosciences)


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


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


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


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    Echelon Biosciences anti pi 3 5 p 2 antibody
    Anti Pi 3 5 P 2 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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    Echelon Biosciences anti pi 3 5 p 2
    Anti Pi 3 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Echelon Biosciences mouse anti pi 3 5 p 2 antibody
    (A) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. PI(3,5)P 2 (red) was detected by immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of the relative intensity of PI(3,5)P 2 from (A) (n = 20). (C) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. PIKfyve, MTM1, and PRV gE were detected by immunoblotting. (D) PK-15 cells were mock-infected or infected with PRV HN1201 (MOI = 0.1) and treated with YM-201636 (10 μM) for 24 hours. PI(3,5)P 2 in the lysosomal fraction was detected by lipid dot-blot analysis. The fraction was analyzed by immunoblotting with an antibody against NPC1 (lysosome marker). (E) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. The colocalization of PI(3,5)P 2 (red) with LAMP1 (lysosomal marker, blue) was detected by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of colocalization of PI(3,5)P 2 with LAMP1 from (E) (n = 40). (G) PK-15 cells were treated with vehicle, YM-201636 (10 μM), or ML-SI1 (10 μM) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (H) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (I) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were incubated with PRV HN1201 (MOI = 0.1), vehicle, or poly-lysine (50 μg/mL) as indicated at 4°C for 2 hours, and then temperature-shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (J) Schematic representation of the rapamycin-inducible heterodimerization system used to recruit MTM1 to the lysosomal membrane. Rab7 is a lysosome-specific Rab protein. MTM1 is a PI 3 phosphatase that can convert PI(3,5)P 2 and PI(3)P into PI(5)P and PI, respectively. (K) HeLa cells were cotransfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. The subcellular localization of mCherry-FRB-MTM1 and BFP-FKBP-Rab7 was observed by fluorescence microscopy. Scale bar: 10 μm. (L) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. Intracellular Ca 2+ levels were detected using Fluo-3 staining (5 μM). Scale bar: 10 μm. (M) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes and infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (N) Mcoln1 +/+ and Mcoln1 -/- MEF cells were transfected with TRPML1-WT or TRPML1-7Q for 24 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). Data are expressed as the mean ± SD of 3 independent experiments. P -values were determined by Student’s t -test. P < 0.05 was considered statistically significant.
    Mouse Anti Pi 3 5 P 2 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
    https://www.bioz.com/result/mouse anti pi 3 5 p 2 antibody/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti pi 3 5 p 2 antibody - by Bioz Stars, 2024-06
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    91
    Echelon Biosciences anti pi 4 5 p 2
    (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.
    Anti Pi 4 5 P 2, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti pi 4 5 p 2/product/Echelon Biosciences
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pi 4 5 p 2 - by Bioz Stars, 2024-06
    91/100 stars
      Buy from Supplier

    86
    Echelon Biosciences anti pi 3 5 p 2 antibody
    (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.
    Anti Pi 3 5 P 2 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
    https://www.bioz.com/result/anti pi 3 5 p 2 antibody/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti pi 3 5 p 2 antibody - by Bioz Stars, 2024-06
    86/100 stars
      Buy from Supplier

    Image Search Results


    (A) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. PI(3,5)P 2 (red) was detected by immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of the relative intensity of PI(3,5)P 2 from (A) (n = 20). (C) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. PIKfyve, MTM1, and PRV gE were detected by immunoblotting. (D) PK-15 cells were mock-infected or infected with PRV HN1201 (MOI = 0.1) and treated with YM-201636 (10 μM) for 24 hours. PI(3,5)P 2 in the lysosomal fraction was detected by lipid dot-blot analysis. The fraction was analyzed by immunoblotting with an antibody against NPC1 (lysosome marker). (E) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. The colocalization of PI(3,5)P 2 (red) with LAMP1 (lysosomal marker, blue) was detected by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of colocalization of PI(3,5)P 2 with LAMP1 from (E) (n = 40). (G) PK-15 cells were treated with vehicle, YM-201636 (10 μM), or ML-SI1 (10 μM) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (H) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (I) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were incubated with PRV HN1201 (MOI = 0.1), vehicle, or poly-lysine (50 μg/mL) as indicated at 4°C for 2 hours, and then temperature-shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (J) Schematic representation of the rapamycin-inducible heterodimerization system used to recruit MTM1 to the lysosomal membrane. Rab7 is a lysosome-specific Rab protein. MTM1 is a PI 3 phosphatase that can convert PI(3,5)P 2 and PI(3)P into PI(5)P and PI, respectively. (K) HeLa cells were cotransfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. The subcellular localization of mCherry-FRB-MTM1 and BFP-FKBP-Rab7 was observed by fluorescence microscopy. Scale bar: 10 μm. (L) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. Intracellular Ca 2+ levels were detected using Fluo-3 staining (5 μM). Scale bar: 10 μm. (M) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes and infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (N) Mcoln1 +/+ and Mcoln1 -/- MEF cells were transfected with TRPML1-WT or TRPML1-7Q for 24 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). Data are expressed as the mean ± SD of 3 independent experiments. P -values were determined by Student’s t -test. P < 0.05 was considered statistically significant.

    Journal: PLOS Pathogens

    Article Title: Pseudorabies virus inhibits progesterone-induced inactivation of TRPML1 to facilitate viral entry

    doi: 10.1371/journal.ppat.1011956

    Figure Lengend Snippet: (A) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. PI(3,5)P 2 (red) was detected by immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of the relative intensity of PI(3,5)P 2 from (A) (n = 20). (C) PK-15 cells were infected with PRV HN1201 (MOI = 0.1) for 0–24 hours. PIKfyve, MTM1, and PRV gE were detected by immunoblotting. (D) PK-15 cells were mock-infected or infected with PRV HN1201 (MOI = 0.1) and treated with YM-201636 (10 μM) for 24 hours. PI(3,5)P 2 in the lysosomal fraction was detected by lipid dot-blot analysis. The fraction was analyzed by immunoblotting with an antibody against NPC1 (lysosome marker). (E) PK-15 cells were mock-infected or infected with PRV-GFP (MOI = 0.1) for 24 hours. The colocalization of PI(3,5)P 2 (red) with LAMP1 (lysosomal marker, blue) was detected by immunofluorescence analysis. Scale bar: 10 μm. (F) Quantification of colocalization of PI(3,5)P 2 with LAMP1 from (E) (n = 40). (G) PK-15 cells were treated with vehicle, YM-201636 (10 μM), or ML-SI1 (10 μM) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (H) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (I) PK-15 cells were treated with vehicle or poly-lysine (50 μg/mL) for 8 hours. Cells were incubated with PRV HN1201 (MOI = 0.1), vehicle, or poly-lysine (50 μg/mL) as indicated at 4°C for 2 hours, and then temperature-shifted to 37°C for 10 minutes to allow entry. After washing with trypsin (1 mg/mL) to remove residual virions on the PM, viral entry was detected by qRT-PCR analysis of viral genome copy numbers in the cells. (J) Schematic representation of the rapamycin-inducible heterodimerization system used to recruit MTM1 to the lysosomal membrane. Rab7 is a lysosome-specific Rab protein. MTM1 is a PI 3 phosphatase that can convert PI(3,5)P 2 and PI(3)P into PI(5)P and PI, respectively. (K) HeLa cells were cotransfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. The subcellular localization of mCherry-FRB-MTM1 and BFP-FKBP-Rab7 was observed by fluorescence microscopy. Scale bar: 10 μm. (L) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes. Intracellular Ca 2+ levels were detected using Fluo-3 staining (5 μM). Scale bar: 10 μm. (M) HeLa cells were transfected with mCherry-FRB-MTM1 (red) and BFP-FKBP-Rab7 (blue) for 24 hours. Cells were then treated with rapamycin (500 nM) for 20 minutes and infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). (N) Mcoln1 +/+ and Mcoln1 -/- MEF cells were transfected with TRPML1-WT or TRPML1-7Q for 24 hours. Cells were then infected with PRV HN1201 (MOI = 0.1). Intracellular Ca 2+ levels were immediately detected using Fluo-3 staining (5 μM). Data are expressed as the mean ± SD of 3 independent experiments. P -values were determined by Student’s t -test. P < 0.05 was considered statistically significant.

    Article Snippet: The membrane was then blocked in 3% fatty acid-free BSA at room temperature for 1 h and probed with mouse anti-PI(3,5)P 2 antibody (1:1,000 dilution; Echelon).

    Techniques: Infection, Immunofluorescence, Western Blot, Dot Blot, Marker, Staining, Incubation, Quantitative RT-PCR, Membrane, Fluorescence, Microscopy, Transfection

    (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.

    Journal: PLoS Pathogens

    Article Title: Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

    doi: 10.1371/journal.ppat.1008317

    Figure Lengend Snippet: (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.

    Article Snippet: Specific PIP residues were detected using anti-PI(3)P (1:100); Purified anti-PI(3)P IgG, Z-P003 Echelon Biosciencies), anti-PI(4,5)P 2 (1:100; Purified anti-PI(4,5)P 2 IgM, Z-P003 Echelon Biosciencies) and anti-PI(3,4,5)P 3 (1:100; Purified anti-PI(3,4,5)P 3 IgM, Z-P045 Echelon Biosciencies) followed by an anti-mouse antibody coupled to fluorochrome in all three cases (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 594, Cat. Nr. A-11005, Thermo Fischer Scientific or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 488, Cat. Nr. A-11017, Thermo Fischer Scientific).

    Techniques: Binding Assay, Immunodetection

    (A-D) Light microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing Legionella -derived PIP-binding constructs. (A-B) Compared to low 2xFYVE-GFP-expressing cells from the same population, reduction of PI(3)P binding sites in cells highly expressing a regulated encystation-dependent epitope-tagged construct 2xFYVE-GFP (GFP) inhibits uptake of fluid-phase marker Dextran-R. Scale bars: 1 μm. (C-D) Expression levels of PI(4)P-binding epitope-tagged construct GFP-P4C expression (GFP) have no visible impact on Dextran-R signal at PVs of transfected cells. Scale bars: 1 μm. (E) Dextran-R uptake in non-transgenic wild-type cells as negative controls for construct-induced uptake phenotypes. Scale bars: 1 μm (F) Box-plot representing the distribution of cell width (in μm) across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) increase in median cell width with respect to non-transgenic cells is detected for 2xFYVE-GFP- but not GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (G) Box-plot representing the distribution of measured Dextran-R signal intensity across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal intensity, normalized to wild-type cells (100%), is detected for 2xFYVE-GFP- but not for GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (H) A statistically significant (p<0.5) linear correlation exists between Dextran-R signal ( x -axis, intensity_red channel [%]) and 2xFYVE-GFP expression ( y -axis, intensity_green channel [%]) measured across 100 cells. (I) The apparent linear correlation between GFP-P4C expression ( y -axis, intensity_green channel [%]) and Dextran-R signal ( x -axis, intensity_red channel [%]) is not statistically significant (p<0.5). (J) Wide-field microscopy-based immunofluorescence analysis of the impact of neomycin treatment on Dextran-R uptake to deplete PI(4,5)P 2 binding sites in non-transgenic wild-type cells. With respect to non-treated cells (WT; left panel), Dextran-R signal at PVs is visibly impacted in non-transgenic neomycin-treated cells (WT_Neo; right panel). Scale bars: 10 μm for full wide-field image, 1 μm for a single cell. (K) Box-plot representing the distribution of measured Dextran-R signal intensity across 100 wild-type cells, either untreated (WT) or treated with neomycin (WT_Neo). Neomycin treatment causes a statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal. Scale bars: wide-field: 10 μm; single cells: 1 μm. For all images, nuclei are labelled with DAPI (blue). Insets: DIC images.

    Journal: PLoS Pathogens

    Article Title: Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

    doi: 10.1371/journal.ppat.1008317

    Figure Lengend Snippet: (A-D) Light microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing Legionella -derived PIP-binding constructs. (A-B) Compared to low 2xFYVE-GFP-expressing cells from the same population, reduction of PI(3)P binding sites in cells highly expressing a regulated encystation-dependent epitope-tagged construct 2xFYVE-GFP (GFP) inhibits uptake of fluid-phase marker Dextran-R. Scale bars: 1 μm. (C-D) Expression levels of PI(4)P-binding epitope-tagged construct GFP-P4C expression (GFP) have no visible impact on Dextran-R signal at PVs of transfected cells. Scale bars: 1 μm. (E) Dextran-R uptake in non-transgenic wild-type cells as negative controls for construct-induced uptake phenotypes. Scale bars: 1 μm (F) Box-plot representing the distribution of cell width (in μm) across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) increase in median cell width with respect to non-transgenic cells is detected for 2xFYVE-GFP- but not GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (G) Box-plot representing the distribution of measured Dextran-R signal intensity across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal intensity, normalized to wild-type cells (100%), is detected for 2xFYVE-GFP- but not for GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (H) A statistically significant (p<0.5) linear correlation exists between Dextran-R signal ( x -axis, intensity_red channel [%]) and 2xFYVE-GFP expression ( y -axis, intensity_green channel [%]) measured across 100 cells. (I) The apparent linear correlation between GFP-P4C expression ( y -axis, intensity_green channel [%]) and Dextran-R signal ( x -axis, intensity_red channel [%]) is not statistically significant (p<0.5). (J) Wide-field microscopy-based immunofluorescence analysis of the impact of neomycin treatment on Dextran-R uptake to deplete PI(4,5)P 2 binding sites in non-transgenic wild-type cells. With respect to non-treated cells (WT; left panel), Dextran-R signal at PVs is visibly impacted in non-transgenic neomycin-treated cells (WT_Neo; right panel). Scale bars: 10 μm for full wide-field image, 1 μm for a single cell. (K) Box-plot representing the distribution of measured Dextran-R signal intensity across 100 wild-type cells, either untreated (WT) or treated with neomycin (WT_Neo). Neomycin treatment causes a statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal. Scale bars: wide-field: 10 μm; single cells: 1 μm. For all images, nuclei are labelled with DAPI (blue). Insets: DIC images.

    Article Snippet: Specific PIP residues were detected using anti-PI(3)P (1:100); Purified anti-PI(3)P IgG, Z-P003 Echelon Biosciencies), anti-PI(4,5)P 2 (1:100; Purified anti-PI(4,5)P 2 IgM, Z-P003 Echelon Biosciencies) and anti-PI(3,4,5)P 3 (1:100; Purified anti-PI(3,4,5)P 3 IgM, Z-P045 Echelon Biosciencies) followed by an anti-mouse antibody coupled to fluorochrome in all three cases (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 594, Cat. Nr. A-11005, Thermo Fischer Scientific or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 488, Cat. Nr. A-11017, Thermo Fischer Scientific).

    Techniques: Light Microscopy, Immunofluorescence, Transgenic Assay, Expressing, Derivative Assay, Binding Assay, Construct, Marker, Transfection, Microscopy