Fig. 5. TGFß levels are increased in AhR–/– MEF. (A) The amount of TGFß secreted by MEF was analyzed in conditioned medium from AhR+/+ and AhR–/– fibroblasts. The inhibitory effect of TGFß on the proliferation of Mv1Lu mink lung epithelial cells was determined by measuring the rate of [³H]-thymidine incorporation during DNA synthesis. Conditioned medium was obtained after 72 hours of culture in OPTI-MEM medium. In some experiments, 1 µg/ml neutralizing anti-TGFß antibody was added during incubation of the Mv1Lu cells with conditioned medium. To determine total TGFß activity, conditioned media were heated at 80°C for 8 minutes before addition to Mv1Lu cultures. To transform [³H]-thymidine incorporation into TGFß concentration, a standard curve was constructed with known amounts of recombinant TGFß protein. Experiments as the one shown were used to calculate the concentration of active and total TGFß presented in Table 2. (B) Tgfß mRNA expression was determined in AhR+/+ and AhR–/– MEF by northern blot using 10 µg total RNA. The expression of ß-actin was used to normalize for equal loading. The experiments were done in triplicate in at least two different MEF cultures.