Anti phosphorylated p38

RBCs were fixed with 3.7% formaldehyde in PBS (pH 7.4) for 10 min at room temperature, washed in the same buffer and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After washing with cold PBS, samples were incubated for 30 minutes at 37°C with monoclonal or polyclonal antibodies. Monoclonal antibodies: anti-ER-α, anti-ER-β, anti-ERK 1/2, anti-P38, anti-phosphorylated P38 (all Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phosphorylated ERK 1/2 (BD PharMingen, San Diego, CA). Polyclonal antibodies: anti-AKT, anti-phosphorylated AKT (Thr 308)-R (all Santa Cruz Biotechnology), and anti-eNOS (phospho S-1177) (Cambridge, MA). As negative control we used mouse or rabbit IgG1 immunoglobulin isotype (Sigma). Samples were washed thrice in PBS to be then incubated with secondary antibody FITC-conjugated: anti-mouse (Invitrogen, Carlsbad, CA) or anti-rabbit (Invitrogen, Carlsbad, CA). For NO detection, samples were incubated with 4, 5-Diaminofluorescein diacetate (Enzo Life Sciences, Lausen, Switzerland) for 30 min at 37°C and washed three times in PBS. All the samples were recorded with a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, USA) equipped with a 488nm argon laser. At least 20, 000 events were acquired. The median values of fluorescence intensity histograms were used to provide a semi-quantitative analysis.