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p22 phox cdna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher p22 phox cdna
    A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the <t>EROS-NOX2-p22</t> <t>phox</t> -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.
    P22 Phox Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2"

    Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

    Journal: bioRxiv

    doi: 10.1101/2023.09.11.557130

    A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.
    Figure Legend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

    Techniques Used: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

    A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.
    Figure Legend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

    Techniques Used: Cryo-EM Sample Prep, Membrane

    A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.
    Figure Legend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

    Techniques Used:



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    A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the <t>EROS-NOX2-p22</t> <t>phox</t> -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.
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    Image Search Results


    A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

    Journal: bioRxiv

    Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

    doi: 10.1101/2023.09.11.557130

    Figure Lengend Snippet: A. Images from confocal fluorescent microscopy showing the expression of EROS (FITC, green fluorescence) and NOX2 (PE, red fluorescence) on the surface of differentiated HL-60 cells (dHL-60). Colocalization of the two membrane proteins (yellow, merged image) was shown in the lower right panel. Scale bar: 5 μm. B. Colocalization of EROS and NOX2 in transiently transfected COS-7 cells. The cells were cotransfected with NOX2-N-Clover (green) and EROS-C-mRubby2 (red). Confocal microscopy images were taken 24 h after transfection. C. Verification of cell surface expression of EROS and NOX2 in dHL-60 by flow cytometry, using the primary antibodies anti-EROS-FITC and anti-NOX2 (7D5) plus PE-labeled goat anti-mouse secondary antibody for 1-h incubation. D. Size-exclusion chromatography of the EROS-NOX2-p22 phox -7G5 Fab complex on Superose 6. The two major peaks were collected and subjected to SDS-PAGE. E. NOX2 was detected at an expected molecular weight of ∼91 kDa, indicating that the EROS-associated NOX2 is in mature form.

    Article Snippet: COS 91/22 was prepared using the original protocol of Dinauer and coworkers [ ] except that the p22 phox cDNA was expressed in pcDNA3.1-neuromycin vector (Thermo Fisher Scientific).

    Techniques: Microscopy, Expressing, Fluorescence, Membrane, Transfection, Confocal Microscopy, Flow Cytometry, Labeling, Incubation, Size-exclusion Chromatography, SDS Page, Molecular Weight

    A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

    Journal: bioRxiv

    Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

    doi: 10.1101/2023.09.11.557130

    Figure Lengend Snippet: A. The cryo-EM map of the EROS-NOX2-p22 phox -7G5 complex. EROS (blue) and p22 phox (purple) are opposite to each other and both associated with NOX2 (green). There is no direct interaction between EROS and p22 phox . For clarity, the 7G5-Fab dimers are colored differently in pink and gray. The left panel shows a side view of the cryo-EM map, and the middle panel depicts a side view at a 90° rotation to the left panel. The right panel is a bottom view of the complex from an intracellular perspective. The inner heme (orange) is visible in this view. B. Cartoon representation of the EROS structure. There are four helices (H1-H4) and six β strands, with the N terminus buried inside and the C terminal fragment (C165-S187) is disordered (not shown). H1 (I21-Y40) and H2 (W47-Q61) are connected by Loop 1; H3 (L85-F93) is nearly perpendicular to H1 and H2. The C terminal H4 (R147-L161) is tilted relative to plasma membrane and does not form a transmembrane helix. The two longest β-strands are anti-parallel and connected by Loop 2 (V117-G121). C. Topological model of EROS (blue), NOX2 (green) and p22 phox (purple) in plasma membrane. The yellow hexagon labels indicate three glycosylation sites on NOX2. LA-LE corresponds to Loop A to Loop E. DH denotes the dehydrogenase domain of NOX2. PH represents the Pleckstrin Homology domain separated by H1 and H2. L1 and L2 refer to Loop 1 and Loop 2. ECL and ICL stand for extracellular and intracellular loops, respectively.

    Article Snippet: COS 91/22 was prepared using the original protocol of Dinauer and coworkers [ ] except that the p22 phox cDNA was expressed in pcDNA3.1-neuromycin vector (Thermo Fisher Scientific).

    Techniques: Cryo-EM Sample Prep, Membrane

    A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

    Journal: bioRxiv

    Article Title: Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2

    doi: 10.1101/2023.09.11.557130

    Figure Lengend Snippet: A. The 3-D reconstruction of the EROS-NOX2-p22 phox complex, with NOX2 in green, EROS in blue and p22 phox in purple. The heme groups are marked in maroon. B. TM2 of NOX2 in the presence of EROS (green) vs. in its absence (gray, PDB ID: 8GZ3), showing a nearly 79° upward rotational shift (red arrow) of a.a. 67-83 of TM2 (dark green) from its position in the resting state (silver). The atom-to-atom distance of the shifted R80 is 18.6Å. TM6 and EROS are removed from this panel for clarity. C. A 45° horizontal rotation of B showing a 48° backward rotation of a.a. 265-292 (dark green) of TM6 when NOX2 is bound to EROS. TM6 in the resting state (PDB ID: 8GZ3) is depicted in gray, and the same fragment in silver. The distance between the shifted Q292 is 41.9Å, along with a movement of the N terminus of NOX2 towards TM2. D. Top view (extracellular view, left) and bottom view (intracellular view, right) of the superimposed NOX2 structures in EROS-bound (green) and resting (gray) states, with emphasis on the dislocated TM2 and TM6 of NOX2. EROS is marked in blue.

    Article Snippet: COS 91/22 was prepared using the original protocol of Dinauer and coworkers [ ] except that the p22 phox cDNA was expressed in pcDNA3.1-neuromycin vector (Thermo Fisher Scientific).

    Techniques:

    Optimized production of NOX2/p22 phox liposomes using the cell-free expression system. Notes: ( A ) Effect of magnesium and potassium concentrations on cell-free production of p22 phox protein. In vitro expression was performed in 96-well plates with a volume of 50 µL. ( B ) Effect of lipid composition on the expression of p22 phox subunit. Total protein fraction was obtained after the cell-free expression reaction, while the proteoliposome fraction was separated after a discontinuous sucrose gradient to separate them from liposomes and aggregated proteins. ( C ) Effect of the reaction time variation on NOX2 and p22 phox expression. Reactions were carried out at 30°C for 2 h, 4 h, 6 h or 16 h, in batch format (100 µL) and separated by SDS-PAGE on a 15% gel. P22 phox and NOX2 detection bands are indicated with stars. ( D ) Effect of the variation of iron and hemin concentration on protein expression. In vitro expression was performed in 96-well plates with a volume of 50 µL. In all experiments, monoclonal anti-His HRP-conjugated antibody and anti-NOX2 antibodies (clone 44.1) were used for the detection of p22 phox and NOX2, respectively. Optimal concentrations were indicated. * Indicates the location of NOX2 and p22 phox . Abbreviations: NTPs, nucleotide triphosphates; PLs, proteoliposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Journal: International Journal of Nanomedicine

    Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

    doi: 10.2147/IJN.S128611

    Figure Lengend Snippet: Optimized production of NOX2/p22 phox liposomes using the cell-free expression system. Notes: ( A ) Effect of magnesium and potassium concentrations on cell-free production of p22 phox protein. In vitro expression was performed in 96-well plates with a volume of 50 µL. ( B ) Effect of lipid composition on the expression of p22 phox subunit. Total protein fraction was obtained after the cell-free expression reaction, while the proteoliposome fraction was separated after a discontinuous sucrose gradient to separate them from liposomes and aggregated proteins. ( C ) Effect of the reaction time variation on NOX2 and p22 phox expression. Reactions were carried out at 30°C for 2 h, 4 h, 6 h or 16 h, in batch format (100 µL) and separated by SDS-PAGE on a 15% gel. P22 phox and NOX2 detection bands are indicated with stars. ( D ) Effect of the variation of iron and hemin concentration on protein expression. In vitro expression was performed in 96-well plates with a volume of 50 µL. In all experiments, monoclonal anti-His HRP-conjugated antibody and anti-NOX2 antibodies (clone 44.1) were used for the detection of p22 phox and NOX2, respectively. Optimal concentrations were indicated. * Indicates the location of NOX2 and p22 phox . Abbreviations: NTPs, nucleotide triphosphates; PLs, proteoliposomes; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    Article Snippet: Then, the R199Q mutant NOX2 cDNA and p22 phox cDNA sequence were subcloned by Proteogenix (Schiltigheim, France) into a specific plasmid optimized by Synthelis SAS.

    Techniques: Expressing, In Vitro, SDS Page, Concentration Assay, Polyacrylamide Gel Electrophoresis

    In vitro physicochemical characterization of NOX2/p22 phox and negative liposomes. Notes: ( A ) Particle size distribution of NOX2/p22 phox and negative liposomes by DLS. ( B ) Coomassie blue staining of NOX2/p22 phox and negative liposomes (The * and ▲ indicate the location of NOX2 and p22 phox respectively) and Western blot analysis of NOX2/p22 phox and negative liposomes using monoclonal antibodies against NOX2 and p22 phox . ( C ) Dithionite-reduced minus-oxidized spectra of NOX2/p22 phox and negative liposomes. Abbreviations: DLS, dynamic light scattering; OD, optical density; PLs, proteoliposomes.

    Journal: International Journal of Nanomedicine

    Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

    doi: 10.2147/IJN.S128611

    Figure Lengend Snippet: In vitro physicochemical characterization of NOX2/p22 phox and negative liposomes. Notes: ( A ) Particle size distribution of NOX2/p22 phox and negative liposomes by DLS. ( B ) Coomassie blue staining of NOX2/p22 phox and negative liposomes (The * and ▲ indicate the location of NOX2 and p22 phox respectively) and Western blot analysis of NOX2/p22 phox and negative liposomes using monoclonal antibodies against NOX2 and p22 phox . ( C ) Dithionite-reduced minus-oxidized spectra of NOX2/p22 phox and negative liposomes. Abbreviations: DLS, dynamic light scattering; OD, optical density; PLs, proteoliposomes.

    Article Snippet: Then, the R199Q mutant NOX2 cDNA and p22 phox cDNA sequence were subcloned by Proteogenix (Schiltigheim, France) into a specific plasmid optimized by Synthelis SAS.

    Techniques: In Vitro, Staining, Western Blot

    In vitro NADPH oxidase activity of NOX2/p22 phox and negative liposomes. Notes: ( A ) Representative results of the NADPH oxidase activity of NOX2/p22 phox (8 pmol cytochrome b 558 ) and negative liposomes in a cell-free system assay in the presence of the recombinant p47 phox , p67 phox and Rac (1 µM), arachidonic acid (400 µM) and NBT (100 µM), stimulated or not with NADPH (200 µM). ( B ) NADPH oxidase activity was expressed in moles of superoxide O 2 •− produced/s/mol of heme and measured before (total activity) or after SOD addition (SOD non-inhibitable activity). Cell-free system assay was performed in the same experimental conditions as ( A ). Abbreviations: DPI, diphenyleneiodonium; NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; OD, optical density; O 2 •− , superoxide anion; SOD, superoxide dismutase; PLs, proteoliposomes.

    Journal: International Journal of Nanomedicine

    Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

    doi: 10.2147/IJN.S128611

    Figure Lengend Snippet: In vitro NADPH oxidase activity of NOX2/p22 phox and negative liposomes. Notes: ( A ) Representative results of the NADPH oxidase activity of NOX2/p22 phox (8 pmol cytochrome b 558 ) and negative liposomes in a cell-free system assay in the presence of the recombinant p47 phox , p67 phox and Rac (1 µM), arachidonic acid (400 µM) and NBT (100 µM), stimulated or not with NADPH (200 µM). ( B ) NADPH oxidase activity was expressed in moles of superoxide O 2 •− produced/s/mol of heme and measured before (total activity) or after SOD addition (SOD non-inhibitable activity). Cell-free system assay was performed in the same experimental conditions as ( A ). Abbreviations: DPI, diphenyleneiodonium; NADPH, nicotinamide adenine dinucleotide phosphate; NBT, nitroblue tetrazolium; OD, optical density; O 2 •− , superoxide anion; SOD, superoxide dismutase; PLs, proteoliposomes.

    Article Snippet: Then, the R199Q mutant NOX2 cDNA and p22 phox cDNA sequence were subcloned by Proteogenix (Schiltigheim, France) into a specific plasmid optimized by Synthelis SAS.

    Techniques: In Vitro, Activity Assay, Recombinant, Produced

    Analysis of the membrane delivery of NOX2 and p22 phox subunits in X 0 -CGD iPSC-derived macrophages after NOX2/p22 phox liposome treatment. Notes: ( A ) Flow cytometry analysis of NOX2 and p22 phox expression using monoclonal antibodies in WT and untreated X 0 -CGD macrophages (black curve), and X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox (red curve) or negative (green curve) liposomes. Isotype controls are represented by gray-filled curves. MFIs were indicated for each condition, and the shift of fluorescence was calculated as the percentage of increased fluorescence compared to untreated macrophages. ( B ) Confocal microscopy images showing the staining of NOX2 subunit with 7D5 antibody and AF488-conjugated secondary antibody (green) in WT and X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox or negative liposomes. Nuclei were counterstained with Hoechst 33258 (blue); scale bars =20 µm. The same observations were obtained in at least two experiments. Abbreviations: CGD, chronic granulomatous disease; iPSC, induced pluripotent stem cell; MFIs, mean fluorescence intensities; MΦ, macrophages; WT, wild type; X 0 -CGD, X 0 -linked CGD; XCGD, X-linked CGD.

    Journal: International Journal of Nanomedicine

    Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

    doi: 10.2147/IJN.S128611

    Figure Lengend Snippet: Analysis of the membrane delivery of NOX2 and p22 phox subunits in X 0 -CGD iPSC-derived macrophages after NOX2/p22 phox liposome treatment. Notes: ( A ) Flow cytometry analysis of NOX2 and p22 phox expression using monoclonal antibodies in WT and untreated X 0 -CGD macrophages (black curve), and X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox (red curve) or negative (green curve) liposomes. Isotype controls are represented by gray-filled curves. MFIs were indicated for each condition, and the shift of fluorescence was calculated as the percentage of increased fluorescence compared to untreated macrophages. ( B ) Confocal microscopy images showing the staining of NOX2 subunit with 7D5 antibody and AF488-conjugated secondary antibody (green) in WT and X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox or negative liposomes. Nuclei were counterstained with Hoechst 33258 (blue); scale bars =20 µm. The same observations were obtained in at least two experiments. Abbreviations: CGD, chronic granulomatous disease; iPSC, induced pluripotent stem cell; MFIs, mean fluorescence intensities; MΦ, macrophages; WT, wild type; X 0 -CGD, X 0 -linked CGD; XCGD, X-linked CGD.

    Article Snippet: Then, the R199Q mutant NOX2 cDNA and p22 phox cDNA sequence were subcloned by Proteogenix (Schiltigheim, France) into a specific plasmid optimized by Synthelis SAS.

    Techniques: Derivative Assay, Flow Cytometry, Expressing, Fluorescence, Confocal Microscopy, Staining

    Location of NOX2 in liposome-treated X 0 -CGD iPSC-derived macrophages after C . albicans phagocytosis. Notes: ( A ) Confocal microscopy images showing the staining of NOX2 subunit with 7D5 antibody and AF488-conjugated secondary antibody (green) in X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox or negative liposomes and then for 4 h with C . albicans strain at an MOI of 3:1. ( B ) Z-stack images (top to bottom of cell) of an NOX2/p22 phox liposome-treated X 0 -CGD macrophage incubated for 4 h with C . albicans strain at an MOI of 3:1. Nuclei were counterstained with Hoechst 33258 (blue); scale bars =20 µm in ( A ) and 10 µm in ( B ). The same observations were obtained in at least two experiments. Abbreviations: C. albicans; Candida albicans , CGD, chronic granulomatous disease; iPSC, induced pluripotent stem cell; MOI, multiplicity of infection; X 0 -CGD, X 0 -linked CGD; XCGD, X-linked CGD.

    Journal: International Journal of Nanomedicine

    Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

    doi: 10.2147/IJN.S128611

    Figure Lengend Snippet: Location of NOX2 in liposome-treated X 0 -CGD iPSC-derived macrophages after C . albicans phagocytosis. Notes: ( A ) Confocal microscopy images showing the staining of NOX2 subunit with 7D5 antibody and AF488-conjugated secondary antibody (green) in X 0 -CGD macrophages treated for 8 h with NOX2/p22 phox or negative liposomes and then for 4 h with C . albicans strain at an MOI of 3:1. ( B ) Z-stack images (top to bottom of cell) of an NOX2/p22 phox liposome-treated X 0 -CGD macrophage incubated for 4 h with C . albicans strain at an MOI of 3:1. Nuclei were counterstained with Hoechst 33258 (blue); scale bars =20 µm in ( A ) and 10 µm in ( B ). The same observations were obtained in at least two experiments. Abbreviations: C. albicans; Candida albicans , CGD, chronic granulomatous disease; iPSC, induced pluripotent stem cell; MOI, multiplicity of infection; X 0 -CGD, X 0 -linked CGD; XCGD, X-linked CGD.

    Article Snippet: Then, the R199Q mutant NOX2 cDNA and p22 phox cDNA sequence were subcloned by Proteogenix (Schiltigheim, France) into a specific plasmid optimized by Synthelis SAS.

    Techniques: Derivative Assay, Confocal Microscopy, Staining, Incubation, Infection

    Analysis of in cellulo toxicity and NADPH oxidase activity restoration in X 0 -CGD iPSC-derived macrophages. Notes: ( A ) Viability of CGDX 0 iPSC-derived macrophages after 8, 12 and 24 h of treatment. Untreated cells are used as positive control (100%) and DMSO-treated cells as negative control. Data are expressed as mean ± SEM ( n =3). The differences of viability between the untreated cells and the liposome-treated cells were analyzed using the nonparametric Mann–Whitney test. ( B ) Morphological images of X 0 -CGD iPSC-derived macrophages after 8, 12 and 24 h of treatment with NOX2/p22 phox liposomes or incubated with IMDM only (magnification ×10). ( C ) X 0 -CGD macrophages were treated for 8 h with NOX2/p22 phox or negative liposomes. Then, WT and X 0 -CGD macrophages were stimulated with PMA (resting; scale bars =50 µm). Abbreviations: CGD, chronic granulomatous disease; DMSO, dimethylsulfoxide; IMDM, Iscove’s Modified Dulbecco’s Medium; iPSC, induced pluripotent stem cell; NADPH, nicotinamide adenine dinucleotide phosphate; ns, nonsignificant; PMA, phorbolmyristate-acetate; SEM, standard error of the mean; WT, wild type; X 0 -CGD, X 0 -linked CGD.

    Journal: International Journal of Nanomedicine

    Article Title: Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

    doi: 10.2147/IJN.S128611

    Figure Lengend Snippet: Analysis of in cellulo toxicity and NADPH oxidase activity restoration in X 0 -CGD iPSC-derived macrophages. Notes: ( A ) Viability of CGDX 0 iPSC-derived macrophages after 8, 12 and 24 h of treatment. Untreated cells are used as positive control (100%) and DMSO-treated cells as negative control. Data are expressed as mean ± SEM ( n =3). The differences of viability between the untreated cells and the liposome-treated cells were analyzed using the nonparametric Mann–Whitney test. ( B ) Morphological images of X 0 -CGD iPSC-derived macrophages after 8, 12 and 24 h of treatment with NOX2/p22 phox liposomes or incubated with IMDM only (magnification ×10). ( C ) X 0 -CGD macrophages were treated for 8 h with NOX2/p22 phox or negative liposomes. Then, WT and X 0 -CGD macrophages were stimulated with PMA (resting; scale bars =50 µm). Abbreviations: CGD, chronic granulomatous disease; DMSO, dimethylsulfoxide; IMDM, Iscove’s Modified Dulbecco’s Medium; iPSC, induced pluripotent stem cell; NADPH, nicotinamide adenine dinucleotide phosphate; ns, nonsignificant; PMA, phorbolmyristate-acetate; SEM, standard error of the mean; WT, wild type; X 0 -CGD, X 0 -linked CGD.

    Article Snippet: Then, the R199Q mutant NOX2 cDNA and p22 phox cDNA sequence were subcloned by Proteogenix (Schiltigheim, France) into a specific plasmid optimized by Synthelis SAS.

    Techniques: Activity Assay, Derivative Assay, Positive Control, Negative Control, MANN-WHITNEY, Incubation, Modification