2.7 Western blot analysis of AKT and p47phoxphosphorylation
The phosphorylation of AKT and p47phox was analysed in
cell pellets from (HS). Cells were suspended in a 2× lysis buffer (5 mM
EDTA, 0.15 mol NaCl, 0.1 mol TRIS, pH 8.0, 1% triton and 10 μg/ml of
protease and phosphatase inhibitors cocktail (Thermo Fisher Scientific,
USA). Equal amounts of protein (30 μg/lane) were solubilized in a 2X
Leammli sample buffer, separated on a 10-12% SDS-polyacrylamide gel and
then electrotransferred to nitrocellulose membranes. After blocking with
Bovine Serum Albumin (BSA, 5%; Sigma Aldrich, USA) the membranes were
incubated overnight at 4°C with rabbit polyclonal anti-p-p47phox (Abcam,
UK), anti-p-AKT and anti-β-Actin antibody (Santa Cruz Biotechnology,
USA) and subsequently with secondary antibody (1:3000; Bio-Rad, USA).
The immune complexes were detected by enhanced chemiluminescence
substrate (ECL Substrates, Bio-Rad, USA). Densitometric analysis of the
bands was performed using Image J software.