Secondary anti rabbit igg

The penile tissue was homogenized in RIPA buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1% Nonidet p-40, 0.5% sodium deoxycholate, 0.1% SDS. The homogenate was separated at 14,000×g for 20 minutes at 4℃ through a centrifuge. After centrifugation, the supernatant was separated and preserved at -80℃. Through the Bradford protein assay, the protein concentration was confirmed. The proteins were diluted in SDS electrophoresis sample buffer, separated on a 6% or 8% SDS-PAGE gel, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Nonspecific binding was blocked using a mixture of Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 5% skim milk for 1 hour at room temperature. And then, the membrane was incubated overnight with rabbit anti-nNOS (diluted 1:500; Abcam) and rabbit anti-eNOS (diluted 1:1,000; Invitrogen, Grand Island, NY, USA) in TBS-T and 5% skim milk at 4℃. The membrane was washed 3 times for 10 minutes using TBS-T. And, we incubated for 1 hour with secondary anti-rabbit IgG (diluted 1:5,000; Invitrogen) conjugated with horseradish peroxidase. Immunoreactivity was confirmed with increased chemiluminescent autoradiography (ECL kit; GE Healthcare, Amersham, UK), according to the manufacturer’s instructions. The immunoreactivity assessments was normalized to beta-actin loading control and evaluate by densitometry using ImageJ.

Total proteins were extracted from 14‐day‐old plants by incubating homogenized samples in sample buffer (20 mm Tris–HCl [pH 6.8], 3% β‐mercaptoethanol, 2.5% SDS, 10% sucrose) with cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland) for 1.5 h at room temperature. After removing debris by centrifugation, the indicated amount of proteins was separated by SDS‐PAGE. For RpoB detection we used the same antibody as described for ptChIP‐seq and a secondary anti‐rabbit IgG conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA, catalog PI314), visualized using chemiluminescence reagents (ECL Prime Western Blotting Detection Reagent, Amersham) and blue films (Kodak, Rochester, NY, USA).