Phosphor erk p erk

Alginate, LPS, PMB, FITC-phalloidin, FITC-LPS and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin were purchased from Hyclone (Logan, UT, USA). Foetal bovine serum (FBS) was obtained from Biological Industries (Beit-Haemek, Israel). Anti-mouse TLR4, MD2, MyD88, F4/80 and Ly6G antibodies were from purchased Abcam (Cambridge, UK). Antibodies against Akt, phosphor-Akt (p-Akt), NF-κB p65, IκB, phosphor-IκB (p-IκB), mTOR, phosphor-mTOR (p-mTOR), p70 S6 kinase (p70 S6K), phosphor-p70 S6K (p-p70 S6K), p38, phosphor-p38 (p-p38), JNK, phosphor-JNK (p-JNK), ERK, phosphor-ERK (p-ERK) and iNOS were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin, -β-tubulin, -GAPDH and -Lamin B1 antibodies were purchased from Proteintech (Hubei, China). Inhibitors LY 294002, SB 20358, SP 600125 and PD 98059 were obtained from Selleck (Shanghai, China) and TAK-242 and rapamycin from Invitrogen (Carlsbad, CA, USA). The other chemicals were all purchased from Macklin Biochemical Technology (Shanghai, China).

Total protein was extracted from bMECs using a total protein extraction kit (BioChain, US) and the protein concentrations were determined by bicinchoninic acid protein assay kit (BioChain, US). All cells were respectively pretreated with Na₂SeO₃ (2, 4, and 8 μM) or left untreated for 12 h and infected with S. aureus (MOI = 1:1) for 0.5 h. Cell lysates were prepared as described previously [2 (link)]. Proteins were loaded into 10% SDS polyacrylamide gel for electrophoresis, and transferred using sodium phosphate buffer to polyvinylidene fluoride membrane metastasis (Merck Millipore, Germany). The membrane was then hybridized with specific antibodies. Antibodies included β-actin, phosphor-IκBα (p-IκBα), phospho-p65 (p-p65), phosphor-p38 (p-p38), and phosphor-Erk (p-Erk) (Cell Signaling Technology, US).