Western Blot Analysis of Signaling Pathways

Stimulated cells were lysed by boiling in 2× SDS-PAGE sample buffer [28 (link)]. To analyse mice infected with C. septicum, muscle and splenic tissues were homogenised in lysis buffer (PBS containing 10% (v/v) Triton-X100) with added protease inhibitor tablets (leupeptin, aprotinin, pepstatin) (Roche, Basel, Switzerland) for 30 min on ice (one tablet in 50 mL of lysis buffer). Lysates were clarified by centrifugation at 12,000× g for 10 min at 4 °C, and supernatants were collected for subsequent Western blotting. Protein concentration was determined using a BCA assay (Pierce, Rockford, IL, USA) and 150 μg of protein was loaded per well. Samples were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Whatman, Kent, UK). Membranes then were probed using antibodies for phospho-ERK, phospho-p38, phospho-JNK, ERK, JNK, p38, phospho c-Raf (ser259) or β-tubulin (Cell Signalling Technology, Danvers, MA, USA). Membranes were stripped in 25 mM Tris-HCl buffer pH 6.5, containing 10% (w/v) SDS and 50 mM β-mercaptoethanol and re-probed or duplicate samples were separated by electrophoresis to assay for protein loading.