LPS induces CD40 expression in a dose- and time-dependent manner. (A) RAW264.7 cells were treated with medium or varying concentrations of LPS (0.1-1000 ng/mL) or IFN-γ (10 ng/mL) for 8 hours, then total RNA was isolated and analyzed by ribonuclease protection assay (RPA) for CD40, TNF-α, and GAPDH mRNA. The basal level of the untreated sample was set as 1.0, and fold induction on LPS or IFN-γ treatment was compared with that. (B) RAW264.7 cells were treated in the absence or presence of LPS or IFN-γ 36 hours, then stained with either anti-CD40 or isotype-matched control antibody. Cells were subjected to fluorescence-activated cell sorting (FACS) analysis. Samples were analyzed by measuring MFI. Fold change in CD40 MFI value was calculated and shown as the mean ± SD of 3 experiments. (C) RAW264.7 and EOC13 cells were treated with 10 ng/mL LPS for up to 48 hours, then RNA was isolated and subjected to RPA analysis for CD40 and GAPDH mRNA. Fold induction on LPS treatment was calculated as in panel A. (D) RAW264.7 cells were treated in the absence or presence of LPS (10 ng/mL) for up to 48 hours. CD40 protein expression was analyzed by FACS analysis. Fold change of LPS-induced CD40 MFI value was calculated and shown as the mean ± SD of 3 experiments. (E) RAW264.7 cells were treated in the absence or presence of LPS (10 ng/mL) for up to 48 hours. Protein lysates were prepared and subjected to immunoblotting with anti-CD40 antibody, then stripped, and reprobed with antiactin antibody as a loading control. UN indicates untreated. Representative of 3 experiments.