The effect of TGFβ on LPS-induced KC mRNA expression is time- and protein synthesis–dependent. (A) RAW264.7 cells were treated or not with TGFβ (20 ng/mL) for 3 hours before adding LPS (10 ng/mL) and/or TGFβ for a further 1 or 3 hours of incubation. Total RNA was prepared and levels of chemokine mRNA were determined as described in the legend to Figure 1. (B) RAW264.7 cells were cultured in the presence of TGFβ (20 ng/mL) for 3 hours and then washed and placed in culture medium without TGFβ for the indicated times prior to the addition of LPS for 3 hours. KC mRNA levels were determined as above. Controls included no treatment (UT), LPS alone for 3 hours, or LPS and TGFβ at the same time for 3 hours. (C) RAW264.7 cells were treated or not with TGFβ (20 ng/mL) in the presence or absence of CHX (10 μg/mL) for 3 hours. Cultures were washed and fresh medium without TGFβ or CHX was added, and the cells were stimulated with LPS for an additional 3 hours prior to analysis of KC mRNA levels. (D) RAW264.7 cells were treated with LPS (10 ng/mL) alone or with TGFβ (20 ng/mL) for 3 hours in the presence of neutralizing antibody against IL-10 as indicated. Total RNA was prepared and analyzed for KC and GAPDH mRNA levels by Northern hybridization. Similar results were obtained in 3 separate experiments.