p22 phox cdna  (Thermo Fisher)

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

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

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: