NF-κB and STAT-1α signaling pathways are activated by LPS. (A) RAW264.7 cells were incubated in the absence or presence of LPS (10 ng/mL) for up to 4 hours. Protein lysates were prepared and subjected to immunoblotting with anti–phospho-IKK-α/β^{Ser180/Ser181}, anti–phospho-IκBα^Ser32, and anti–phospho-NF-κB p65^Ser536, stripped and reprobed with anti–NF-κB p65 and antiactin as loading controls. (B) RAW264.7 cells were incubated in medium or LPS (10 ng/mL) for up to 4 hours, lysed, and assayed for expression of IRF-3 protein. Actin protein expression was used as a loading control. (C) RAW264.7 or EOC13 cells were treated with LPS (10 ng/mL) for up to 12 hours, then RNA was isolated and subjected to RPA analysis for IFN-β and GAPDH mRNA expression. (D) RAW264.7 cells were treated with LPS (10 ng/mL) for up to 12 hours, then supernatants were collected and subjected to ELISA analysis for IFN-β protein expression. Data are presented as the mean ± SD of 3 experiments. (E) RAW264.7 cells were incubated in medium or LPS (10 ng/mL) for up to 4 hours, then cell lysates were prepared and subjected to immunoblotting with anti–phospho-STAT-1α^Tyr701 and anti–phospho-STAT-1α^Ser727, stripped, and reprobed with anti–STAT-1α and antiactin as loading controls. (F) RAW264.7 cells were treated with medium or LPS (10 ng/mL) in the absence or presence of 10 μg/mL isotype antibody or IFN-β–neutralizing antibody for 4 hours. Cell lysates were prepared and subjected to immunoblotting with anti–phospho-STAT-1α^Tyr701. Total STAT-1α and actin protein expression were used as loading controls. Representative of 3 experiments.