Phospho c raf ser259

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.

The following antibodies and reagents were purchased: CDK4 (#12790), CDK6 (#13331), cyclin D1 (#55506), cyclin E (#20808), p21 (#2947), p27 (#3686), PARP (#9532), caspase-9 (#9504), cleaved-caspase-3 (#9664), AKT (#4691), phospho-AKT (Ser473) (#4060), phospho-PDK1 (Ser241) (#3438), PTEN (#9188), phospho-PTEN (Ser380) (#9551) and phospho-c-raf (Ser259) (#9421) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Bcl-2 (ab32124), Bcl-XL (ab32370), Bax (ab32503) and cytochrome c (ab133504) antibodies were purchased from abcam. GAPDH (60004-1-Ig), Survivin (10508-1-AP), XIAP (66800-1-Ig), COXIV (11242-1-AP) and PCNA (60097-1-Ig) antibodies were purchased from Proteintech Group (Chicago, IL, USA). Rhein (R7269), Oxaliplatin (O9512) and N-acetyl-L-cysteine (NAC, A7250) were obtained from Sigma-Aldrich (St. Louis, MO, USA). DCFH-DA (S0033) and JC-1 (C2006) were purchased from Beyotime (Haimen, China). Z-DEVD-FMK (S7312), z-LEHD-FMK (S7313) and z-VAD-FMK (S8102) were purchased from Selleck (Houston, TX, USA). LY294002 (HY-10108) and 740Y-P (HY-P0175) were purchased from MCE (Monmouth, NJ, USA).

Monoclonal antibodies used in this study were directed against phospho-MET (Tyr1234/1235) (Cell Signaling Technology), MET (clone D1C2, Cell Signaling Technology), phospho-EGFR (Tyr845) (Cell Signaling Technology), phospho-ALK (Tyr1064) (Cell Signaling Technology), phospho-ATM (Ser1981) (Cell Signaling Technology), phospho-c-Raf (Ser259) (Cell Signaling Technology), phospho-AKT (Ser473) (Cell Signaling Technology), phospho-MAPK (Thr202/Tyr204) (Cell Signaling Technology), phospho-H2AX (Ser139) (Upstate), Ki67 (Cell Signaling Technology) and ß-actin (Millipore Chemicon).