Anti p s1177 enos

Cells were transfected and harvested in lysis buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5 % NP-40, MiniComplete protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). Equal protein amounts were loaded on 8 % gels and resolved by electrophoresis. Samples were transferred to PVDF membranes and labeled overnight with primary antibody: anti-β-actin (0.2 μg.mL⁻¹, Santa Cruz); anti-total-eNOS (0.2 μg.mL⁻¹, BD Biosciences); anti-p(S1177)-eNOS (0.2 μg.mL⁻¹, BD Biosciences); or anti-Piezo1 (0.3 μg.mL⁻¹, Proteintech). Horse radish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch) and SuperSignal Femto detection reagent (Perbio Science) were used for visualization. Densities of protein bands were quantified by ImageJ software. Catalogue information for antibodies: anti-β-actin (0.2 μg.mL⁻¹, Clone C4, sc-47778; Santa Cruz); anti-total-eNOS (0.2 μg.mL⁻¹, Clone 3/eNOS/NOS Type III, Cat. #610297; BD Biosciences); anti-p(S1177)-eNOS (0.2 μg.mL⁻¹, Cat. #612392; BD Biosciences); or anti-Piezo1 (0.3 μg.mL⁻¹, Proteintech).