Lipopolysaccharide lps from salmonella minnesota r595

PD0325901 (0.1 μM; pre‐treatment for 10 min) was produced by the Division of Signal Transduction Therapy (DSTT), University of Dundee. VX‐745 (1 μM; pre‐treatment for 1 h) was purchased from Selleckchem. C34 (10 μM; pre‐treatment for 1 h) was from MedChemExpress. Leupeptin (100 μg/ml; pre‐treatment for 1 h) and bafilomycin A1 (1 μM; pre‐treatment for 15 min) were purchased from Sigma‐Aldrich. Lipopolysaccharide (LPS) from Salmonella minnesota R595 was from Enzo Life Sciences.

RAW 264.7 mouse macrophage cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum(FBS), 1 × glutamine, and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, referred to as complete DMEM (cDMEM). For stable isotope labeling by amino acids in cell culture (SILAC), the cells were cultured in DMEM for SILAC purchased from Thermo Fisher Scientific (Waltham, MA, USA), supplemented with 10% FBS, 1 × glutamine, 20 mM HEPES buffer, and isotopically labeled lysine and arginine purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). The cells were cultured in the labeled media for five passages prior to analysis to allow for > 95% incorporation of the labeled amino acids. Lipopolysaccharide (LPS) from Salmonella minnesota R595 was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA).