P22phox

For co-immunoprecipitation assay, cells were washed with PBS and harvested in a cell lysis buffer (30 mM Tris, pH 8.0, 10 mM NaCl,5 mM EDTA, 10 g/l polyoxyethylene-8-lauryl ether, 1 mM 0-phenanthroline,1 mM iodoacetamide, 10 mM NaF, 5 mM orthovanadate, and 10 mM sodium pyrophosphate). After being precleared using 20 μl of protein A/G PLUS-agarose (Santa Cruz Biotechnology Inc.), cell lysates were incubated with the first protein-specific antiserum (e.g., anti-p47phox) overnight at 4°C and then were incubated with 20 μl of protein A/G PLUS-agarose for additional 2 h with rotation. All immunoprecipitated samples were washed with lysis buffer three times and subjected to SDS-PAGE, followed by immunoblot with the second antibody (e.g., NOX2 or AIP1). The chemiluminescence was detected using ECL kit (Merck Millipore).
For immunoblot in artery and culture cells, protein was extracted from homogenized tissues or cells in lysis buffer, and boiled in SDS sample buffer for 10 min. Equal amounts of protein per sample were separated by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories). Antibodies against AIP1 were described previously (Zhang et al., 2003 (link)). Primary antibodies used included AIP1 (Invitrogen), NOX1, NOX2, NOX4, NOX5 (Abcam), p47phox, p22phox (Cell Signaling Technology), FLAG, HA and GAPDH (Sigma).