Ep1 4

Western blotting was carried out as previously described (Newson et al., 2014 (link)). Briefly, cells from peritoneal washouts or ex vivo culture were lysed in RIPA buffer with protease inhibitors (both Sigma) and the protein concentration determined by Bradford assay (Bio-Rad). Ten micrograms of protein were separated by SDS-PAGE (National Diagnostics). Separated proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon; Millipore) and incubated with COX-1, COX-2, mPGES-1, mPGE-2, EP1-4 (Cayman Chemical), β-actin, and GAPDH (Sigma) overnight in block buffer (Tris-HCL, 1% Tween-20, 1% BSA [Sigma], and 5% nonfat milk [Marvel]). Blots were washed and incubated with HRP-conjugated antibodies (Santa Cruz Biotechnology) for 1 hr at room temperature in blocking buffer. Specific proteins were visualized by enhanced chemiluminescence (ECL) hyperfilm.

Total proteins were extracted in (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% Na-deoxycholate, 0.1% SDS) supplemented with protease inhibitors. Protein concentration was determined using BCA protein assay (Sigma). Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore, St Quentin-Yvelines, France) and revealed with ECL (Millipore). Antibodies that recognize actin (Millipore), Bax (BD Pharmingen, San Jose, CA, USA), Bcl-2 (BD Pharmingen), EGFR and pEGFR (Cell Signaling Technology, Denvers, MA, USA), Bad (Cell Signaling), EP1–4 (Cayman, Ann Arbor, MI, USA), caspase 3 (Santa Cruz Biotech, Santa Cruz CA, USA), GFAP (Calbiochem, Darmstadt, Germany), nestin (Millipore, Temecula, CA, USA), olig2 (Abcam, Cambridge, UK) and β-tubulin (Sigma–Aldrich) were used. HRP-conjugated secondary antibodies were from BioRad. The ImageJ64 software was used to quantify Western blot bands. caspase 3 activity was determined using the fluorogenic substrate Ac-DEVD-AMC, as described in [14 (link)]. Note, for all the assays the cut-off limit of caspase 3 activation that induces cell death was determined at > 50 AU/mg protein.