As described previously,10 19 (link)20 (link)21 22 (link) western blot assay was carried out to determine protein expression levels. Briefly, after cavernous tissue samples were homogenized, equal amounts of protein (50 μg in each well) were electrophoresed on Mini Protean TGX gels (7.5% or 12.0%; Bio-Rad, Hercules, CA, USA). They were run at 200 V for 30 min and transferred to polyvinylidene fluoride (PDVF) membrane (Merck Millipore) at 100 V for 60 min. After adding primary antibodies, they were incubated overnight at 4°C. After adding secondary antibody, they were incubated for 60 min. Primary antibodies were as follows: anti-Akt (1:5000, Cell-Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Ser473, 1:1000, Cell-Signaling Technology), anti-eNOS (1:3000, BD Biosciences, San Jose, CA, USA), anti-phospho-eNOS (Ser1177, 1:1000, Cell-Signaling Technology), anti-nNOS (1:2000, Cell-Signaling Technology), anti-phospho-nNOS (Ser1417, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA), anti-phospho-LIMK2 (phospho T505; Thr505, 1:1000, Abcam), anti-LIMK2 (1:2000, Abcam), anti-phospho-cofilin (Ser3, 1:1000, Cell-Signaling Technology), anti-cofilin (1:1000, Cell-Signaling Technology), and anti-fibronectin (1:1000, Abcam). Results were quantified by densitometry and presented as relative density of each protein compared to that of β-actin.