α complete

For western blotting analysis, tissue extracts were prepared by homogenizing mouse hippocampus tissue in T-PER Tissue Protein Extraction Reagent (Thermo Scientific Inc., Waltham, MA, USA) containing protease inhibitor, α-complete (Roche Applied Science, Penzberg, Germany). Fifteen micrograms of each tissue extract sample were adjusted to give a final solution of 60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5% β-mercaptoethanol. The solution was heated at 100ºC for 5 min, electrophoresed through a 10% SDS-polyacrylamide gel, and transferred to polyvinylidine difluoride membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Plexin-A1, Neuropilin-1, COX-2, iNOS, IL-1β, TNF-α and β-actin were detected by their respective antibodies using an enhanced chemiluminescence western blot detection system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Anti-Plexin-A1 antibody (Abcam), anti-Neuropilin-1 antibody (Abcam), anti-COX-2 antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), anti-iNOS antibody (Merck Millipore, Darmstadt, Germany), anti-IL-1β antibody (Santa Cruz Biotechnology), anti-TNF-α antibody (Santa Cruz Biotechnology), and anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA) were used.

For Western blot analysis, tissue extracts were prepared by homogenizing mouse vaginal tissue in T-PER Tissue Protein Extraction Reagent (Thermo Scientific Inc., Waltham, MA, USA) containing a protease inhibitor (α-complete; Roche Applied Science, Penzberg, Germany) and a phosphatase inhibitor (PhosStop; Roche Applied Science). Protein quantification of the tissue extract was performed using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Each sample (15 μg) was prepared in a final solution of 60 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.1% bromophenol blue and 5% β-mercaptoethanol. The sample solution was heated at 100°C for 5 min, electrophoresed through a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Sema4D, plexin-B1 and cleaved caspase-3 were detected with their respective antibodies using an enhanced chemiluminescence or enhanced chemiluminescence-plus western blot detection system in accordance with the manufacturer’s instructions (Amersham Pharmacia Biotech). The antibodies used were anti-CD100/Sema4D (cat.no. 610670; BD Transduction Laboratories, Franklin Lakes, NJ, USA), anti-plexin-B1 (cat.no. sc-28372; Santa Cruz Biotechnology, Inc.) and anti-cleaved caspase-3 (cat.no. #9664 Cell Signaling Technology).