Localization of Nox2 N-terminus using polyclonal antipeptide antibodies.
Nox2/gp91(phox) (where phox is phagocyte oxidase) is the catalytic membrane subunit of the granulocyte NADPH oxidase complex involved in host defence. The current model of membrane topology of Nox2 is based upon the identification of glycosylation sites, of regions that interact with the regulatory cytosolic factors and of the epitopes recognized by antibodies. So far, the localization of the N-terminus of Nox2 was only speculative. In order to clarify this localization, we raised a polyclonal antiserum against the N-terminal sequence M(1)GNWVAVNEGL(11). Purified antibodies recognize the mature protein as a broad band at 91 kDa (glycosylated form) or a band at 55 kDa after deglycosylation. Immunocytochemistry and flow-cytometry analysis show a strong binding of the anti-N-terminal antibodies to differentiated HL60 cells and neutrophils respectively, after permeabilization only. The N-terminus of Nox2 is therefore present in the mature protein and is located to the cytoplasmic side of the plasma membrane.
Paclet
Marie-Hélène
MH
GREPI EA 2938, Laboratoire d'Enzymologie/DBPC, CHU Albert Michallon, BP 217, 38043 Grenoble Cédex 9, France.
Henderson
Lydia M
LM
Campion
Yannick
Y
Morel
Françoise
F
Dagher
Marie-Claire
MC
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D002454:Cell Differentiation; D003593:Cytoplasm / Q000737:chemistry; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000032:analysis* / Q000737:chemistry*; D009504:Neutrophils / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis
D000906:Antibodies; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases