P thr 495 enos

The proteins obtained from the cell lysates (10 μg) were resolved on 8.5% SDS-PAGE gels and transferred to PVDF membranes. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies were incubated for 90 minutes at room temperature. The membranes were stripped after detection of the phosphorylated eNOS in order to detect total eNOS levels. Antibodies and dilutions were used as follows: eNOS (BD Transduction Lab, Allschwil, Switzerland, 610297, 1:5000), p-Thr-495 eNOS (Cell signaling, Leiden, The Netherlands, 9574S, 1:1000), and β-actin-HRP (Abcam, Cambridge, UK, ab49900, 1:50,000). β-actin was used as a loading control. The amount of proteins was detected using a chemiluminiscence kit (Millipore, Burlington, MA, USA) in the ChemiDoc System (Bio-Rad). For the eNOS dimer and monomer quantification, non-boiled samples in reducing conditions and containing 20 μg of total protein were resolved in a 6% SDS-PAGE at 4 °C and low voltage (60 V). Then, the proteins were transferred to a PVDF membrane and Western blot analysis for eNOS was performed as described above.

Western blotting was executed as previously described [13 (link)]. Briefly, cell lysates were blotted with antibodies specific to p-ERK (Cell Signaling, Danvers, MA, USA), ERK (Cell Signaling), p-Akt (Cell Signaling), Akt (Cell Signaling), endothelial nitric oxide synthetase (eNOS, Cell Signaling), p-Ser1177 eNOS (Cell Signaling), p-Ser633 eNOS (Bioss Antibodies Inc., Woburn, MA, USA), p-Thr495 eNOS (Cell Signaling), protein kinase Cε (PKCε, Santa Cruz), p-Cav-1 (BD Biosciences, Franklin Lakes, NJ, USA), Cav-1 (BD Biosciences), IκB (Sigma), and β-actin (Santa Cruz).