7.4), NP-40 (1%), sodium deoxycholate (0.5%) and SDS (0.1%). Samples were
centrifuged at 5,000 × g for 10 min (4ºC). Forty micrograms of protein
were separated by electrophoresis on a 10% polyacrylamide gel, and transferred
onto a nitrocellulose membrane. Skimmed milk 5% diluted in Tris-buffered saline
solution with Tween 20 was used to block nonspecific binding sites (1 h at
24ºC). Membranes were then incubated overnight at 4ºC with the following primary
antibodies: p-eNOS (Ser1177) (diluted 1:1000, 9571, Cell Signaling,
Danvers, MA, USA), p-eNOS (Thr495) (diluted 1:1000, 9574, Cell
Signaling), total eNOS (diluted 1:1000, 9572, Cell Signaling), P-protein kinase
B (P-Akt) (Ser473) (diluted 1:1000, 4058, Cell Signaling) and total
Akt (diluted 1:1000, 9272, Cell Signaling). Membranes were then incubated with
secondary antibodies for 90 min at room temperature. Signals were revealed with
chemiluminescence and quantified densitometrically. The results are expressed as
the non-phospho/total proteins ratio.