FIGURE 7.

The inhibitory effects of TGF-β1 on surface FcεRI expression are reversible and do not enhance the rate of FcεRI degradation. A, BMMC were cultured for 3 days with or without TGF-β1. Half the TGF-β1-stimulated cells were washed and replated in medium without TGF-β1 on day 3. Over the following 48 h, samples were assessed for surface FcεRI expression by flow cytometry. Percent inhibition of surface FcεRI expression was determined using cultures never receiving TGF-β1, as described in Fig. 1. Data are the mean and SE (too small to be visualized) of eight BMMC populations. B, BMMC were cultured for 3 days in IL-3 and SCF with or without TGF-β1. On day 3, cycloheximide (CHX) or DMSO was added to the cultures for 2–16 h. Using DMSO-treated cells as a control, the CHX-mediated decrease in surface FcεRI expression on cells cultured with or without TGF-β1 was assessed by flow cytometry. Data shown are the mean and SE of 12 samples. C, Cells were cultured and treated with CHX or DMSO as described in B. Total cell lysates from 1 million cells were subjected to SDS-PAGE and Western blotting to detect FcεRI β-chain expression. The percent decrease in expression obtained from three experiments is shown below the respective bands. D, BMMC were cultured in IL-3 with or without TGF-β1 for 3 days. Cells were starved of cysteine and methionine (control), then incubated with ³⁵S-labeled cysteine and methionine for 20 min. FcεRI β-chain and actin were sequentially immunoprecipitated and resolved by gel electrophoresis as described in Materials and Methods.