Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-?B/MAPK Signaling Pathways

Park, Jeong-In; Song, Kyung-Hee; Jung, Seung-Youn; Ahn, Jiyeon; Hwang, Sang-Gu; Kim, Joon; Kim, Eun Ho; Song, Jie-Young

Total proteins from RAW 264.7 cells were extracted in TNN buffer (50 mM Tris-Cl, pH 7.4; 1% NP-40; 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitors (1 mmol/ L phenylmethylsulfonyl fluoride [PMSF], 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 1 mmol/L Na 3 VO 4 ) and quantified using the Bradford method. Protein samples (15 mg) were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking nonspecific antibody binding sites, the membrane was incubated overnight at 4 C with mouse monoclonal antibodies against iNOS, extracellular signal-regulated kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, IkB-a, pIkB-a, and GAPDH. After incubation with peroxidase-conjugated secondary antibodies at 37 C for 1 hour, the protein bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Biosciences, Pittsburgh, Pennsylvania) and detected using the Amersham Imager 680 (GE Healthcare Biosciences). The relative levels of protein expression were calculated with reference to the levels of GAPDH.