Anti phospho cofilin

Male, 5-week-old ICR mice were purchased from Damul Science (Daejeon, Korea). Ten-week-old OVX mice were purchased from Central Lab. Animal Inc. (Seoul, Korea). Mice were kept in a temperature (22 °C–24 °C) and humidity (55%–60%) controlled environment with a 12 h light/dark cycle. All experiments were performed in accordance with guidelines for animal experimentation of the Institute Committee of Wonkwang University. Eupatilin was obtained from DONG-A PHARM (Seoul, Korea). Soluble, recombinant human M-CSF and human RANKL were obtained from PeproTech EC, Ltd. (London, UK). Anti-Akt, anti-phospho-Akt, anti-GSK3β, anti-phospho-GSK3β, anti-IκB, anti-phospho-IκB, anti-Cofilin, anti-phospho-Cofilin, anti-p38, anti-phospho-p38, anti-ERK, anti-phospho-ERK, anti-JNK, and anti-phospho-JNK antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-c-Fos, and anti-NFATc1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A monoclonal β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), α-minimum essential medium (α-MEM), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture.

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.

The tissue was homogenized in radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Biotechnology, Shanghai, China) and incubated on ice for 30 min. The supernatant following centrifugation (at 15,000 × g for 15 min at 4°C) was used for western blotting. Western blotting was performed as described previously (25 (link)). Blots were incubated overnight with the following primary antibodies: Anti-podoplanin (rabbit polyclonal antibody; 1:300; cat. no. 251419; Abbiotec, San Diego, CA, USA), anti-β-tubulin (mouse monoclonal antibody; 1:5,000; cat. no. AT0003; CMCTAG, Inc., Milwaukee, WI, USA) and anti-cofilin (rabbit monoclonal antibody; 1:1,000; cat. no. 5175, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-cofilin (rabbit monoclonal antibody; 1:1,000; cat. no. 3313; Cell Signaling Technology), anti-ERM (rabbit monoclonal antibody; 1:1,000; cat. no. 3142; Cell Signaling Technology), anti-phospho-ERM (rabbit monoclonal antibody; 1:1,000; cat. no. 3726; Cell Signaling Technology), anti-MLC-2 (rabbit monoclonal antibody; 1:1,000; cat. no. 8505; Cell Signaling Technology) and anti-phospho-MLC-2 (1:1,000; cat. no. 3671; Cell Signaling Technology). Western blots were visualized using an electrophoretic gel imaging system (Shanghai, China).

For analysis of cofilin and p-cofilin protein, neurons were lysed in RIPA buffer with protease and protease inhibitor cocktail (Roche). Proteins was separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Anti-PAK1 (1:500 dilution, #SC-166887, Santa Cruz Biotechnology), anti-Phospho-PAK1 (1:500 dilution, #2605P, Cell Signaling Technology), and anti-Phospho-cofilin (1:1,000 dilution, #3311, Cell Signaling) antibodies were used. Western blotting was performed using horseradish peroxidase-conjugated immunoglobulin G as a secondary antibody and the ECL system for detection.

Western blot analyses were performed as described previously.11 (link)12 (link)13 (link) Primary antibodies included anti-ROCK1 (1:2000; Cell-Signaling Technology, Danvers, MA, USA), anti-phospho-LIMK2 (phospho T505, 1:1000; Abcam), anti-LIMK2 (1:2000; Abcam), anti-phospho-Cofilin (1:500; Cell-Signaling Technology), and anti-Cofilin (1:1000; Cell-Signaling Technology). Densitometry results were normalized to β-actin expressions.