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


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    Echelon Biosciences ptdins 4 p
    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 <t>-PtdIns(4)P</t> (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
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    1) Product Images from "Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis"

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103097

    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
    Figure Legend Snippet: Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Techniques Used: Inhibition, Activity Assay, Staining, Purification, Phosphatase Assay, Incubation

    The transient increase in plasma membrane PtdIns(4)P levels in response to H 2 O 2 generated in EGF-activated cells. (A) Western blot analysis for the detection of oxidized Synj2 using cysteine biotinylation assay in A549 cells expressing Myc-tagged full-length Synj2 during EGF (200 ng/ml) activation (Left). Quantification of oxidized Synj2 bands. Error bars represent the SEM from three independent experiments (Student's t-test; *, p < 0.05; **, p < 0.01) (Right). (B) A transient increase in the plasma membrane PtdIns(4)P levels. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. Serum-starved A549 cells were preincubated with DPI (5 μM), a Nox inhibitor, or catalase (2 mg/ml). Scale bars, 20 μm; 4 μm in magnification. (C) Quantification of relative plasma membrane PtdIns(4)P fluorescence in live A549 cells during EGF stimulation. The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19).
    Figure Legend Snippet: The transient increase in plasma membrane PtdIns(4)P levels in response to H 2 O 2 generated in EGF-activated cells. (A) Western blot analysis for the detection of oxidized Synj2 using cysteine biotinylation assay in A549 cells expressing Myc-tagged full-length Synj2 during EGF (200 ng/ml) activation (Left). Quantification of oxidized Synj2 bands. Error bars represent the SEM from three independent experiments (Student's t-test; *, p < 0.05; **, p < 0.01) (Right). (B) A transient increase in the plasma membrane PtdIns(4)P levels. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. Serum-starved A549 cells were preincubated with DPI (5 μM), a Nox inhibitor, or catalase (2 mg/ml). Scale bars, 20 μm; 4 μm in magnification. (C) Quantification of relative plasma membrane PtdIns(4)P fluorescence in live A549 cells during EGF stimulation. The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19).

    Techniques Used: Membrane, Generated, Western Blot, Cell Surface Biotinylation Assay, Expressing, Activation Assay, Fluorescence

    Control of the plasma-membrane PtdIns(4)P levels by Synj2. (A) Transient increase in the plasma membrane PtdIns(4)P levels induced by Synj2. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. siRNA oligo against Synj2 and/or siRNA-resistant Myc-Synj2 was transfected into the cells. Scale bars, 20 μm; 4 μm in magnification. (B) Quantification of the relative plasma-membrane PtdIns(4)P fluorescence in (A). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; **, p < 0.01; n = 45). (C) Immunoblot of Synj2 in the lysates of control and Synj2 knock-down cells.
    Figure Legend Snippet: Control of the plasma-membrane PtdIns(4)P levels by Synj2. (A) Transient increase in the plasma membrane PtdIns(4)P levels induced by Synj2. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. siRNA oligo against Synj2 and/or siRNA-resistant Myc-Synj2 was transfected into the cells. Scale bars, 20 μm; 4 μm in magnification. (B) Quantification of the relative plasma-membrane PtdIns(4)P fluorescence in (A). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; **, p < 0.01; n = 45). (C) Immunoblot of Synj2 in the lysates of control and Synj2 knock-down cells.

    Techniques Used: Membrane, Expressing, Transfection, Fluorescence, Western Blot

    Control of receptor-mediated endocytosis by plasma-membrane H 2 O 2 . (A) Analysis of plasma membrane H 2 O 2 levels in live A549 cells expressing plasma membrane-targeted Hyper (Hyper-Ras) and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The H 2 O 2 levels were measured using relative emission activated by 488-nm and 405-nm lasers (Em 488 /Em 405 ). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 18). (B) Analysis of the plasma membrane PtdIns(4)P levels in live A549 cells expressing FAPP1-PH (mut)-GFP and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 23). (C) Inhibition of EGFR endocytosis through the ectopic expression of Cat-PM. Representative confocal images of fluorescent EGF-555 (EGF conjugated with Alexa 555 dye) endocytosed in control or Cat-PM-expressing cells. Scale bars, 20 μm; 2 μm in magnification. The graph shows the relative intensity of EGF-555 spots 30 min after EGF treatment. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19). Western blot analysis shows Cat-PM expression. (D) Increase in EGFR endocytosis induced by PtdIns(4)P. The amount of endocytosed EGF is increased in cells treated with soluble fluorescent PtdIns(4)P (10 μM). Representative confocal images show PtdIns(4)P (green) and EGF-555 (red) 30 min after EGF treatment. Scale bars, 20 μm; 2 μm in magnification. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 13).
    Figure Legend Snippet: Control of receptor-mediated endocytosis by plasma-membrane H 2 O 2 . (A) Analysis of plasma membrane H 2 O 2 levels in live A549 cells expressing plasma membrane-targeted Hyper (Hyper-Ras) and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The H 2 O 2 levels were measured using relative emission activated by 488-nm and 405-nm lasers (Em 488 /Em 405 ). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 18). (B) Analysis of the plasma membrane PtdIns(4)P levels in live A549 cells expressing FAPP1-PH (mut)-GFP and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 23). (C) Inhibition of EGFR endocytosis through the ectopic expression of Cat-PM. Representative confocal images of fluorescent EGF-555 (EGF conjugated with Alexa 555 dye) endocytosed in control or Cat-PM-expressing cells. Scale bars, 20 μm; 2 μm in magnification. The graph shows the relative intensity of EGF-555 spots 30 min after EGF treatment. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19). Western blot analysis shows Cat-PM expression. (D) Increase in EGFR endocytosis induced by PtdIns(4)P. The amount of endocytosed EGF is increased in cells treated with soluble fluorescent PtdIns(4)P (10 μM). Representative confocal images show PtdIns(4)P (green) and EGF-555 (red) 30 min after EGF treatment. Scale bars, 20 μm; 2 μm in magnification. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 13).

    Techniques Used: Membrane, Expressing, Fluorescence, Inhibition, Western Blot

    Model structures of Synj and the Sac1 domain/PtdIns(4)P complex. (A) Overall structure of the Sac1 domain. The N -terminal and catalytic subdomains are colored in gray and pale cyan, respectively. The P -segment is highlighted in magenta with labeled secondary structural elements (β16 and α10). (B–C) Binding mode of PtdIns(4)P into the active site of Sac1 domain. (B) PtdIns(4)P (yellow) and the interacting residues of both the Sac1 domain (pale cyan) and the P -segment (magenta) are represented as sticks. The 4-P phosphate, 4’-, and 5′-carbon atoms of the inositol ring are marked as 4-P, 4′C, and 5′C, respectively. Gray dotted lines indicate polar interactions. Oxygen, nitrogen, sulfur, and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. (C) Electrostatic potential surfaces of the Sac1 domain. Blue, red, and white colors indicate positively-charged, negatively-charged, and hydrophobic surfaces, respectively. The P -segment is highlighted with a white dotted circle. (D) Overall structure of Synj. For clarity, only the 5-phosphatase domain (green) and the Sac1 domain (pale cyan) are depicted, with emphasis on the active sites of each domain highlighted in gray shading. The PtdIns(3,4,5)P 3 (blue stick) and a magnesium ion (blue sphere) in the 5-phosphatase domain are derived from the superposed structure of the Synj1/PtdIns(3,4,5)P 3 complex (PDB code: 7A17). The PtdIns(4)P (yellow stick) in the Sac1 domain is derived from the docked complex structure.
    Figure Legend Snippet: Model structures of Synj and the Sac1 domain/PtdIns(4)P complex. (A) Overall structure of the Sac1 domain. The N -terminal and catalytic subdomains are colored in gray and pale cyan, respectively. The P -segment is highlighted in magenta with labeled secondary structural elements (β16 and α10). (B–C) Binding mode of PtdIns(4)P into the active site of Sac1 domain. (B) PtdIns(4)P (yellow) and the interacting residues of both the Sac1 domain (pale cyan) and the P -segment (magenta) are represented as sticks. The 4-P phosphate, 4’-, and 5′-carbon atoms of the inositol ring are marked as 4-P, 4′C, and 5′C, respectively. Gray dotted lines indicate polar interactions. Oxygen, nitrogen, sulfur, and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. (C) Electrostatic potential surfaces of the Sac1 domain. Blue, red, and white colors indicate positively-charged, negatively-charged, and hydrophobic surfaces, respectively. The P -segment is highlighted with a white dotted circle. (D) Overall structure of Synj. For clarity, only the 5-phosphatase domain (green) and the Sac1 domain (pale cyan) are depicted, with emphasis on the active sites of each domain highlighted in gray shading. The PtdIns(3,4,5)P 3 (blue stick) and a magnesium ion (blue sphere) in the 5-phosphatase domain are derived from the superposed structure of the Synj1/PtdIns(3,4,5)P 3 complex (PDB code: 7A17). The PtdIns(4)P (yellow stick) in the Sac1 domain is derived from the docked complex structure.

    Techniques Used: Labeling, Binding Assay, Derivative Assay

    Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.
    Figure Legend Snippet: Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Techniques Used: Activation Assay, Membrane

    ptdins 4 p  (Echelon Biosciences)


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    Echelon Biosciences ptdins 4 p
    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 <t>-PtdIns(4)P</t> (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
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    1) Product Images from "Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis"

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103097

    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
    Figure Legend Snippet: Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Techniques Used: Inhibition, Activity Assay, Staining, Purification, Phosphatase Assay, Incubation

    The transient increase in plasma membrane PtdIns(4)P levels in response to H 2 O 2 generated in EGF-activated cells. (A) Western blot analysis for the detection of oxidized Synj2 using cysteine biotinylation assay in A549 cells expressing Myc-tagged full-length Synj2 during EGF (200 ng/ml) activation (Left). Quantification of oxidized Synj2 bands. Error bars represent the SEM from three independent experiments (Student's t-test; *, p < 0.05; **, p < 0.01) (Right). (B) A transient increase in the plasma membrane PtdIns(4)P levels. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. Serum-starved A549 cells were preincubated with DPI (5 μM), a Nox inhibitor, or catalase (2 mg/ml). Scale bars, 20 μm; 4 μm in magnification. (C) Quantification of relative plasma membrane PtdIns(4)P fluorescence in live A549 cells during EGF stimulation. The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19).
    Figure Legend Snippet: The transient increase in plasma membrane PtdIns(4)P levels in response to H 2 O 2 generated in EGF-activated cells. (A) Western blot analysis for the detection of oxidized Synj2 using cysteine biotinylation assay in A549 cells expressing Myc-tagged full-length Synj2 during EGF (200 ng/ml) activation (Left). Quantification of oxidized Synj2 bands. Error bars represent the SEM from three independent experiments (Student's t-test; *, p < 0.05; **, p < 0.01) (Right). (B) A transient increase in the plasma membrane PtdIns(4)P levels. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. Serum-starved A549 cells were preincubated with DPI (5 μM), a Nox inhibitor, or catalase (2 mg/ml). Scale bars, 20 μm; 4 μm in magnification. (C) Quantification of relative plasma membrane PtdIns(4)P fluorescence in live A549 cells during EGF stimulation. The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19).

    Techniques Used: Membrane, Generated, Western Blot, Cell Surface Biotinylation Assay, Expressing, Activation Assay, Fluorescence

    Control of the plasma-membrane PtdIns(4)P levels by Synj2. (A) Transient increase in the plasma membrane PtdIns(4)P levels induced by Synj2. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. siRNA oligo against Synj2 and/or siRNA-resistant Myc-Synj2 was transfected into the cells. Scale bars, 20 μm; 4 μm in magnification. (B) Quantification of the relative plasma-membrane PtdIns(4)P fluorescence in (A). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; **, p < 0.01; n = 45). (C) Immunoblot of Synj2 in the lysates of control and Synj2 knock-down cells.
    Figure Legend Snippet: Control of the plasma-membrane PtdIns(4)P levels by Synj2. (A) Transient increase in the plasma membrane PtdIns(4)P levels induced by Synj2. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. siRNA oligo against Synj2 and/or siRNA-resistant Myc-Synj2 was transfected into the cells. Scale bars, 20 μm; 4 μm in magnification. (B) Quantification of the relative plasma-membrane PtdIns(4)P fluorescence in (A). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; **, p < 0.01; n = 45). (C) Immunoblot of Synj2 in the lysates of control and Synj2 knock-down cells.

    Techniques Used: Membrane, Expressing, Transfection, Fluorescence, Western Blot

    Control of receptor-mediated endocytosis by plasma-membrane H 2 O 2 . (A) Analysis of plasma membrane H 2 O 2 levels in live A549 cells expressing plasma membrane-targeted Hyper (Hyper-Ras) and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The H 2 O 2 levels were measured using relative emission activated by 488-nm and 405-nm lasers (Em 488 /Em 405 ). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 18). (B) Analysis of the plasma membrane PtdIns(4)P levels in live A549 cells expressing FAPP1-PH (mut)-GFP and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 23). (C) Inhibition of EGFR endocytosis through the ectopic expression of Cat-PM. Representative confocal images of fluorescent EGF-555 (EGF conjugated with Alexa 555 dye) endocytosed in control or Cat-PM-expressing cells. Scale bars, 20 μm; 2 μm in magnification. The graph shows the relative intensity of EGF-555 spots 30 min after EGF treatment. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19). Western blot analysis shows Cat-PM expression. (D) Increase in EGFR endocytosis induced by PtdIns(4)P. The amount of endocytosed EGF is increased in cells treated with soluble fluorescent PtdIns(4)P (10 μM). Representative confocal images show PtdIns(4)P (green) and EGF-555 (red) 30 min after EGF treatment. Scale bars, 20 μm; 2 μm in magnification. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 13).
    Figure Legend Snippet: Control of receptor-mediated endocytosis by plasma-membrane H 2 O 2 . (A) Analysis of plasma membrane H 2 O 2 levels in live A549 cells expressing plasma membrane-targeted Hyper (Hyper-Ras) and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The H 2 O 2 levels were measured using relative emission activated by 488-nm and 405-nm lasers (Em 488 /Em 405 ). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 18). (B) Analysis of the plasma membrane PtdIns(4)P levels in live A549 cells expressing FAPP1-PH (mut)-GFP and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 23). (C) Inhibition of EGFR endocytosis through the ectopic expression of Cat-PM. Representative confocal images of fluorescent EGF-555 (EGF conjugated with Alexa 555 dye) endocytosed in control or Cat-PM-expressing cells. Scale bars, 20 μm; 2 μm in magnification. The graph shows the relative intensity of EGF-555 spots 30 min after EGF treatment. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19). Western blot analysis shows Cat-PM expression. (D) Increase in EGFR endocytosis induced by PtdIns(4)P. The amount of endocytosed EGF is increased in cells treated with soluble fluorescent PtdIns(4)P (10 μM). Representative confocal images show PtdIns(4)P (green) and EGF-555 (red) 30 min after EGF treatment. Scale bars, 20 μm; 2 μm in magnification. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 13).

    Techniques Used: Membrane, Expressing, Fluorescence, Inhibition, Western Blot

    Model structures of Synj and the Sac1 domain/PtdIns(4)P complex. (A) Overall structure of the Sac1 domain. The N -terminal and catalytic subdomains are colored in gray and pale cyan, respectively. The P -segment is highlighted in magenta with labeled secondary structural elements (β16 and α10). (B–C) Binding mode of PtdIns(4)P into the active site of Sac1 domain. (B) PtdIns(4)P (yellow) and the interacting residues of both the Sac1 domain (pale cyan) and the P -segment (magenta) are represented as sticks. The 4-P phosphate, 4’-, and 5′-carbon atoms of the inositol ring are marked as 4-P, 4′C, and 5′C, respectively. Gray dotted lines indicate polar interactions. Oxygen, nitrogen, sulfur, and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. (C) Electrostatic potential surfaces of the Sac1 domain. Blue, red, and white colors indicate positively-charged, negatively-charged, and hydrophobic surfaces, respectively. The P -segment is highlighted with a white dotted circle. (D) Overall structure of Synj. For clarity, only the 5-phosphatase domain (green) and the Sac1 domain (pale cyan) are depicted, with emphasis on the active sites of each domain highlighted in gray shading. The PtdIns(3,4,5)P 3 (blue stick) and a magnesium ion (blue sphere) in the 5-phosphatase domain are derived from the superposed structure of the Synj1/PtdIns(3,4,5)P 3 complex (PDB code: 7A17). The PtdIns(4)P (yellow stick) in the Sac1 domain is derived from the docked complex structure.
    Figure Legend Snippet: Model structures of Synj and the Sac1 domain/PtdIns(4)P complex. (A) Overall structure of the Sac1 domain. The N -terminal and catalytic subdomains are colored in gray and pale cyan, respectively. The P -segment is highlighted in magenta with labeled secondary structural elements (β16 and α10). (B–C) Binding mode of PtdIns(4)P into the active site of Sac1 domain. (B) PtdIns(4)P (yellow) and the interacting residues of both the Sac1 domain (pale cyan) and the P -segment (magenta) are represented as sticks. The 4-P phosphate, 4’-, and 5′-carbon atoms of the inositol ring are marked as 4-P, 4′C, and 5′C, respectively. Gray dotted lines indicate polar interactions. Oxygen, nitrogen, sulfur, and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. (C) Electrostatic potential surfaces of the Sac1 domain. Blue, red, and white colors indicate positively-charged, negatively-charged, and hydrophobic surfaces, respectively. The P -segment is highlighted with a white dotted circle. (D) Overall structure of Synj. For clarity, only the 5-phosphatase domain (green) and the Sac1 domain (pale cyan) are depicted, with emphasis on the active sites of each domain highlighted in gray shading. The PtdIns(3,4,5)P 3 (blue stick) and a magnesium ion (blue sphere) in the 5-phosphatase domain are derived from the superposed structure of the Synj1/PtdIns(3,4,5)P 3 complex (PDB code: 7A17). The PtdIns(4)P (yellow stick) in the Sac1 domain is derived from the docked complex structure.

    Techniques Used: Labeling, Binding Assay, Derivative Assay

    Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.
    Figure Legend Snippet: Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Techniques Used: Activation Assay, Membrane

    synj with ptdins 4 5 p 2  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences synj with ptdins 4 5 p 2
    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
    Synj With Ptdins 4 5 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 "Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis"

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103097

    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
    Figure Legend Snippet: Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Techniques Used: Inhibition, Activity Assay, Staining, Purification, Phosphatase Assay, Incubation

    Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.
    Figure Legend Snippet: Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Techniques Used: Activation Assay, Membrane

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    Echelon Biosciences d myo phosphatidylinositol 4 5 bisphosphate ptdins 4 5 p2 d sn 1 2 di o octanoylglyceryl
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    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 <t>-PtdIns(4)P</t> (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
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    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).
    Synj With Ptdins 4 5 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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    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Inhibition, Activity Assay, Staining, Purification, Phosphatase Assay, Incubation

    The transient increase in plasma membrane PtdIns(4)P levels in response to H 2 O 2 generated in EGF-activated cells. (A) Western blot analysis for the detection of oxidized Synj2 using cysteine biotinylation assay in A549 cells expressing Myc-tagged full-length Synj2 during EGF (200 ng/ml) activation (Left). Quantification of oxidized Synj2 bands. Error bars represent the SEM from three independent experiments (Student's t-test; *, p < 0.05; **, p < 0.01) (Right). (B) A transient increase in the plasma membrane PtdIns(4)P levels. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. Serum-starved A549 cells were preincubated with DPI (5 μM), a Nox inhibitor, or catalase (2 mg/ml). Scale bars, 20 μm; 4 μm in magnification. (C) Quantification of relative plasma membrane PtdIns(4)P fluorescence in live A549 cells during EGF stimulation. The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19).

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: The transient increase in plasma membrane PtdIns(4)P levels in response to H 2 O 2 generated in EGF-activated cells. (A) Western blot analysis for the detection of oxidized Synj2 using cysteine biotinylation assay in A549 cells expressing Myc-tagged full-length Synj2 during EGF (200 ng/ml) activation (Left). Quantification of oxidized Synj2 bands. Error bars represent the SEM from three independent experiments (Student's t-test; *, p < 0.05; **, p < 0.01) (Right). (B) A transient increase in the plasma membrane PtdIns(4)P levels. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. Serum-starved A549 cells were preincubated with DPI (5 μM), a Nox inhibitor, or catalase (2 mg/ml). Scale bars, 20 μm; 4 μm in magnification. (C) Quantification of relative plasma membrane PtdIns(4)P fluorescence in live A549 cells during EGF stimulation. The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19).

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Membrane, Generated, Western Blot, Cell Surface Biotinylation Assay, Expressing, Activation Assay, Fluorescence

    Control of the plasma-membrane PtdIns(4)P levels by Synj2. (A) Transient increase in the plasma membrane PtdIns(4)P levels induced by Synj2. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. siRNA oligo against Synj2 and/or siRNA-resistant Myc-Synj2 was transfected into the cells. Scale bars, 20 μm; 4 μm in magnification. (B) Quantification of the relative plasma-membrane PtdIns(4)P fluorescence in (A). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; **, p < 0.01; n = 45). (C) Immunoblot of Synj2 in the lysates of control and Synj2 knock-down cells.

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Control of the plasma-membrane PtdIns(4)P levels by Synj2. (A) Transient increase in the plasma membrane PtdIns(4)P levels induced by Synj2. Selected snapshot images of live A549 cells expressing FAPP1-PH (mut)-GFP. siRNA oligo against Synj2 and/or siRNA-resistant Myc-Synj2 was transfected into the cells. Scale bars, 20 μm; 4 μm in magnification. (B) Quantification of the relative plasma-membrane PtdIns(4)P fluorescence in (A). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; **, p < 0.01; n = 45). (C) Immunoblot of Synj2 in the lysates of control and Synj2 knock-down cells.

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Membrane, Expressing, Transfection, Fluorescence, Western Blot

    Control of receptor-mediated endocytosis by plasma-membrane H 2 O 2 . (A) Analysis of plasma membrane H 2 O 2 levels in live A549 cells expressing plasma membrane-targeted Hyper (Hyper-Ras) and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The H 2 O 2 levels were measured using relative emission activated by 488-nm and 405-nm lasers (Em 488 /Em 405 ). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 18). (B) Analysis of the plasma membrane PtdIns(4)P levels in live A549 cells expressing FAPP1-PH (mut)-GFP and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 23). (C) Inhibition of EGFR endocytosis through the ectopic expression of Cat-PM. Representative confocal images of fluorescent EGF-555 (EGF conjugated with Alexa 555 dye) endocytosed in control or Cat-PM-expressing cells. Scale bars, 20 μm; 2 μm in magnification. The graph shows the relative intensity of EGF-555 spots 30 min after EGF treatment. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19). Western blot analysis shows Cat-PM expression. (D) Increase in EGFR endocytosis induced by PtdIns(4)P. The amount of endocytosed EGF is increased in cells treated with soluble fluorescent PtdIns(4)P (10 μM). Representative confocal images show PtdIns(4)P (green) and EGF-555 (red) 30 min after EGF treatment. Scale bars, 20 μm; 2 μm in magnification. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 13).

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Control of receptor-mediated endocytosis by plasma-membrane H 2 O 2 . (A) Analysis of plasma membrane H 2 O 2 levels in live A549 cells expressing plasma membrane-targeted Hyper (Hyper-Ras) and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The H 2 O 2 levels were measured using relative emission activated by 488-nm and 405-nm lasers (Em 488 /Em 405 ). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 18). (B) Analysis of the plasma membrane PtdIns(4)P levels in live A549 cells expressing FAPP1-PH (mut)-GFP and plasma membrane-targeted catalase (Cat-PM), catalase (Cat), or cytosolic catalase (Cat-cyto). The bar graph shows the relative fluorescence intensity at 2 min in EGF-activated A549 cells. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 23). (C) Inhibition of EGFR endocytosis through the ectopic expression of Cat-PM. Representative confocal images of fluorescent EGF-555 (EGF conjugated with Alexa 555 dye) endocytosed in control or Cat-PM-expressing cells. Scale bars, 20 μm; 2 μm in magnification. The graph shows the relative intensity of EGF-555 spots 30 min after EGF treatment. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 19). Western blot analysis shows Cat-PM expression. (D) Increase in EGFR endocytosis induced by PtdIns(4)P. The amount of endocytosed EGF is increased in cells treated with soluble fluorescent PtdIns(4)P (10 μM). Representative confocal images show PtdIns(4)P (green) and EGF-555 (red) 30 min after EGF treatment. Scale bars, 20 μm; 2 μm in magnification. The error bars represent the SEM (Student's t-test; *, p < 0.05; n = 13).

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Membrane, Expressing, Fluorescence, Inhibition, Western Blot

    Model structures of Synj and the Sac1 domain/PtdIns(4)P complex. (A) Overall structure of the Sac1 domain. The N -terminal and catalytic subdomains are colored in gray and pale cyan, respectively. The P -segment is highlighted in magenta with labeled secondary structural elements (β16 and α10). (B–C) Binding mode of PtdIns(4)P into the active site of Sac1 domain. (B) PtdIns(4)P (yellow) and the interacting residues of both the Sac1 domain (pale cyan) and the P -segment (magenta) are represented as sticks. The 4-P phosphate, 4’-, and 5′-carbon atoms of the inositol ring are marked as 4-P, 4′C, and 5′C, respectively. Gray dotted lines indicate polar interactions. Oxygen, nitrogen, sulfur, and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. (C) Electrostatic potential surfaces of the Sac1 domain. Blue, red, and white colors indicate positively-charged, negatively-charged, and hydrophobic surfaces, respectively. The P -segment is highlighted with a white dotted circle. (D) Overall structure of Synj. For clarity, only the 5-phosphatase domain (green) and the Sac1 domain (pale cyan) are depicted, with emphasis on the active sites of each domain highlighted in gray shading. The PtdIns(3,4,5)P 3 (blue stick) and a magnesium ion (blue sphere) in the 5-phosphatase domain are derived from the superposed structure of the Synj1/PtdIns(3,4,5)P 3 complex (PDB code: 7A17). The PtdIns(4)P (yellow stick) in the Sac1 domain is derived from the docked complex structure.

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Model structures of Synj and the Sac1 domain/PtdIns(4)P complex. (A) Overall structure of the Sac1 domain. The N -terminal and catalytic subdomains are colored in gray and pale cyan, respectively. The P -segment is highlighted in magenta with labeled secondary structural elements (β16 and α10). (B–C) Binding mode of PtdIns(4)P into the active site of Sac1 domain. (B) PtdIns(4)P (yellow) and the interacting residues of both the Sac1 domain (pale cyan) and the P -segment (magenta) are represented as sticks. The 4-P phosphate, 4’-, and 5′-carbon atoms of the inositol ring are marked as 4-P, 4′C, and 5′C, respectively. Gray dotted lines indicate polar interactions. Oxygen, nitrogen, sulfur, and phosphorus atoms are colored in red, blue, yellow, and orange, respectively. (C) Electrostatic potential surfaces of the Sac1 domain. Blue, red, and white colors indicate positively-charged, negatively-charged, and hydrophobic surfaces, respectively. The P -segment is highlighted with a white dotted circle. (D) Overall structure of Synj. For clarity, only the 5-phosphatase domain (green) and the Sac1 domain (pale cyan) are depicted, with emphasis on the active sites of each domain highlighted in gray shading. The PtdIns(3,4,5)P 3 (blue stick) and a magnesium ion (blue sphere) in the 5-phosphatase domain are derived from the superposed structure of the Synj1/PtdIns(3,4,5)P 3 complex (PDB code: 7A17). The PtdIns(4)P (yellow stick) in the Sac1 domain is derived from the docked complex structure.

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Labeling, Binding Assay, Derivative Assay

    Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Activation Assay, Membrane

    Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Inhibition of Synj2 4-phosphatase activity by H 2 O 2 . (A) Coomassie blue staining of purified GST-tagged Synj2 4-phosphatase and 5-phosphatase domains. Each lane contained 1 μg of protein. (B) Phosphatase assay of Synj2 4-phosphatase and 5-phosphatase domains using the malachite green method. The purified Synj2 4-phosphatase and 5-phosphatase proteins were incubated with diC 8 -PtdIns(4)P (echelon) and diC 8 -PtdIns(4,5)P 2 (echelon), respectively. ( C –D) Control of Synj2 phosphatase activity by H 2 O 2 . (C) Purified GST-4-phosphatase domain (3 μg) and (D) purified GST-5-phosphatase domain (3 μg) were incubated with 0, 10, 50, and 200 μM H 2 O 2 for 5 min at room temperature, and the samples were then incubated with 2 μg of catalase for 10 min at 37 °C to remove any remaining H 2 O 2 in a tube. The activity was measured using the malachite green method. The results are shown as the mean ± SEM of triplicates (Student's t-test; *, p < 0.05; **, p < 0.01; n = 3 for each experiment).

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Inhibition, Activity Assay, Staining, Purification, Phosphatase Assay, Incubation

    Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Journal: Redox Biology

    Article Title: Control of the signaling role of PtdIns(4)P at the plasma membrane through H 2 O 2 -dependent inactivation of synaptojanin 2 during endocytosis

    doi: 10.1016/j.redox.2024.103097

    Figure Lengend Snippet: Proposed explanation for the consequence of H 2 O 2 -dependent inactivation of Synj during receptor-mediated endocytosis. Conversion of PtdIns(4,5)P 2 to PtdIns(4)P is achieved through the H 2 O 2 -dependent oxidation of the 4-phosphatase domain of the Synj dual phosphatase and is required for receptor-mediated endocytosis (RME) during EGF activation. PI(3)K C2α uses PtdIns(4)P as a substrate to produce PtdIns(3,4)P 2 , which is critical for the formation and maturation of clathrin-coated pits. Complete degradation of PtdIns(4)P to PtdIns by Synj occurs in the endosome with a low level of H 2 O 2 . The membrane in red shows a PI-rich submembrane compartment. See the discussion for details.

    Article Snippet: As PtdIns(4)P is an intermediate product of Synj with PtdIns(4,5)P 2 , we investigated whether PtdIns(4)P directly controls receptor-mediated endocytosis by monitoring the endocytosis EGFs during receptor activation in cells with or without excess PtdIns(4)P. We administered water-soluble fluorescent PtdIns(4)P (Echelon) and shuttle PIP™ carrier (Echelon) to cells and monitored EGF receptor endocytosis.

    Techniques: Activation Assay, Membrane