The effect of TGFβ on LPS-induced KC and MIP-2 gene transcription. (A) TG-elicited macrophages were cultured in medium alone or with LPS (10 ng/mL) in the absence or presence of TGFβ (20 ng/mL) for the indicated times. Nuclei were isolated and used for determination of transcription for KC, MIP-2, and tubulin by nuclear run-on analysis as described in “Materials and methods.” (B) RAW264.7 cells were either untreated or treated with LPS (10 ng/mL) in the presence or absence of TGFβ (20 ng/mL) for the times indicated before the preparation of nuclear extracts. Of each nuclear extract, 5 μg was analyzed for NFκB DNA-binding activity by EMSA using radiolabeled oligonucleotides containing the κ B1 sequence from the KC promoter. (C) RAW264.7 cells were transiently transfected with the indicated constructs, and after 24 hours of rest, the cells were either untreated or stimulated with LPS (10 ng/mL) in the presence or absence of TGFβ (20 ng/mL) for 18 hours before the analysis of CAT protein production by ELISA. Different KC promoter fragments linked to CAT are illustrated schematically. Values presented are the means ± SEM of 3 separate experiments. Similar results were obtained in 2 separate experiments.