Supersignal west pico chemiluminescence detection system

For protein expression analysis, we isolated and collected the ovaries. For adherent cells, we digested the samples with 0.25% trypsin and collected the samples. The ovaries and cells were lysed by Radioimmunoprecipitation assay buffer (RIPA, 50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA), and phenylmethanesulfonyl fluoride (PMSF, Amresco, 0754, USA) was added to the lysate. The polyvinylidene fluoride membranes (PVDF) membrane (Millipore, IPVH00010, USA) was visualized using the SuperSignal West Pico Chemiluminescence Detection System (Thermo, Prod 34,080, USA) [70 (link)].

Protein was extracted from SIRC cells as follows: cells were washed with PBS (pH 7.4), centrifuged at 1000 × g for 3 min, suspended in protein extraction buffer and incubated on ice for 30 min. After sonication, extract was centrifuged at 8,000 × g for 15 min. Protein samples (30 μg/lane) were subjected to SDS-PAGE and, after transfer to nitrocellulose membranes, were incubated as described previously (Lupo et al., 2007 (link); Scuderi et al., 2008 (link)) with antibody against TNF-α, IL-1β, p-cPLA2, cPLA2, COX-2, β-actin overnight at 4°C followed by incubation with horseradish peroxidase conjugated secondary antibody, goat anti-rabbit IgG for IL-1β, p-cPLA2, cPLA2, COX-2 and goat anti-mouse IgG for TNF-α and β-actin. After washing, with TBS-T, protein expression was visualized with the Super Signal West Pico Chemiluminescence detection system (Thermo Scientific, Rockford, IL, USA). β-actin served as the loading control. Bands were analyzed using Image J software (Version1.43, Broken Symmetry Software, Bethesda, MD, USA).