RAW264.7 cells (5 × 104 cells/mL) were incubated with or without RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 10 min and irradiated (10 Gy) using a blood γ irradiator and incubated at 37 °C for 24 h. Cells were then washed twice with PBS. Cells were incubated with or without selleck kinase inhibitor RGSF (2.5 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 10 min and stimulated

with LPS (0.1 μg/mL) for 24 h. Cytokine levels in the culture supernatant were evaluated using an IL-1β ELISA kit following the manufacturer’s protocol (BD, Franklin Lakes, CA, USA). RAW264.7 cells (2 × 106 cells/mL) were transfected with 10 μg plasmid containing NF-κB-Luc, AP-1-Luc, and TK-renilla-Luc using electrophoresis according to the manufacturer’s instructions (Neon Transfection System; Invitrogen, Carlsbad, CA, USA). The cells were used for experiments 24 h after transfection. Luciferase assay was performed using the Luciferase Assay System (Promega, Madison, WI, USA)

as reported previously [14]. After the indicated treatment in RAW264.7 cells was terminated, total proteins were prepared using Pro-prep lysis buffer (iNtRON, Seoul, Korea) according to the manufacturer’s instructions. Concentrations LBH589 of the extracted proteins were determined using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA); 50 μg proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The blots were blocked with Tris-buffered saline and Tween 20 containing 5% skimmed milk (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and probed with primary antibody diluted in 5% bovine serum albumin (Santa Cruz Biotechnology). The immunoblots were incubated with horseradish peroxidase secondary antibody, and antibody binding was visualized using enhanced chemiluminescence (ECL Plus Western Thiamine-diphosphate kinase Blotting Detection Reagent GE Healthcare, Little Chalfont, UK).

Data were represented as the mean ± standard error of the mean (SEM) of at least three independent experiments, performed in triplicate. Student’s t test was carried out to analyze the statistical significance between the groups using SPSS version 18.0 (SPSS, Chicago, IL, USA). A p value < 0.05 was considered statistically significant. To determine whether IR could enhance the NO-producing capability of signals of LPS, RAW264.7 cells were first irradiated with different doses of radiation (0 Gy, 2.5 Gy, 5 Gy, 10 Gy, and 20 Gy; 2.5 Gy/min) and then left without further treatment or exposed to LPS (0.1 μg/mL) for 24 h postirradiation. As shown in Fig. 1A, increased NO production was observed in irradiated cells in response to LPS at doses as low as 2.5 Gy. Meanwhile, treatment with radiation alone did not induce measurable NO production (data not shown). The maximal effect of radiation was observed at 20 Gy.