NOXO1 Overexpression Lysate

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

NOXO1 (Nox organizing protein 1) and NOXA1 (Nox Activating protein 1) are homologs of p47phox and p67phox. p47phox functions in phagocytes as an essential organizing protein mediating the binding of other regulatory proteins during activation of the phagocyte oxidase, and its translocation to the membrane is triggered upon cell activation by hyperphosphorylation, which relieves autoinhibition of SH3 and PX domains. NOXO1 lacks an auto inhibitory region and phosphorylation sites that are present in p47phox. Co-transfection of Nox1, NOXO1 and NOXA1 reconstitutes ROS (reactive oxygen species) generation in HEK 293 cells in the absence of cell stimulation. NOXO1 binds to the phosphatidylinositol (PtdIns) lipids PtdIns 3,5-P2, PtdIns 5-P and PtdIns 4-P. Unlike p47phox, which is located in the cytosol of resting cells and translocates to the plasma membrane where gp91phox is located, NOXO1 co-localizes with Nox1 in the membranes of resting cells. This localization of NOXO1 is dictated by its PX domain, since this domain but not the remainder of the molecule localizes to membranes.

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Array NBL1-13735

• NOXO1 Antibodies
• NOXO1 Lysates
• NOXO1 Proteins