Anti p enos ser1177

Protein extracts were obtained from kidneys using T-PER Mammalian Protein Extraction Reagent (Pierce, Thermo Scientific, Rockford, USA), as indicated by the manufacturer, in the presence of a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich, Milan, Italy). Protein content was determined with bicinchoninic acid protein assays (BCA, Pierce, Euroclone, Milan, Italy). An appropriate amount of protein was run on SDS-PAGE under reducing conditions for immunoblotting. The separated proteins were then semi-dry transferred to a nitrocellulose membrane (Bio-Rad Laboratories) and proteins of interest were revealed with specific antibodies: anti-p-S6 (Ser235/236), anti-S6, anti-p-AKT (Ser473), anti-AKT, anti-p-eNOS (Ser1177), anti-eNOS, anti-COX IV, anti-Cyt c, and anti-Grp78 (all from Cell Signaling, Euroclone, Milan, Italy); anti-SIRT1, anti-Grp75, and anti-Bcl-2 (all from Santa Cruz); and anti-PGC-1α (Abcam, Cambridge, UK) each at 1:1,000 dilution. Anti-β-Actin (1:10,000; Cell Signaling) and anti-Vinculin (1:10,000; Sigma-Aldrich) were used as loading controls. Immunostaining was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse immunoglobulin for 1 h at room temperature (Tedesco et al. 2010 (link)). Amounts of each protein were measured using SuperSignal Substrate (Pierce) and densitometrically quantified with an IMAGEJ software image analyzer.

An additional set of experiments (n = 15 rats) was performed for biochemical analysis. Lungs were quickly rinsed in ice‐cold PBS (pH 7.4) and clamped between steel tongs pre‐cooled with liquid N₂ and stored at ‐80°C until analysis. For Western blotting analyses, frozen lung tissues were homogenized in lysis buffer plus protease inhibitor cocktail (Santa Cruz Biotechnology) and processed as described elsewhere 24, using the following primary antibodies: anti‐p‐eNOS (Ser¹¹⁷⁷, Cell Signalling Technology), anti‐eNOS (Santa Cruz Biotechnology), anti‐p‐Akt (Ser⁴⁷³) and anti‐Akt (both Cell Signaling Technologies). Band intensities were quantified using ImageJ software.