STAT5 regulates bcl-2 mRNA by binding a conserved site in intron 2 of the bcl-2 gene as determined by chromatin immunoprecipitation. (A) Mast cells as in Figure 1 were incubated in IL-3 (5 ng/mL) plus SCF (50 ng/mL), or IL-3 (5 ng/mL) alone (B) for 24 hours. mRNA level of bcl-2, bcl-X_L, cis, or osm was quantitated by RT-PCR. Values obtained from wild-type mast cells were set to 1 (for IL-3 + SCF: osm group, *P = .03; for IL-3 alone: bcl-2 group, *P = .01 and bcl-X_L group, *P = .03; Student t test). Error bars represent SD of the mean. (C) STAT5 was immunoprecipitated from formaldehyde–cross-linked lysates prepared from STAT5ab^null/null mast cells transduced with either wild-type STAT5a (WT) or STAT5aΔN (ΔN). Binding of STAT5 to putative STAT5 consensus binding sites 1 and 2 in intron 2 of the bcl-2 gene was determined by PCR amplification of immunoprecipitated DNA. Nonspecific binding was assayed by immunoprecipitating STAT5 from non–cross-linked lysates and rIgG isotype-control immunoprecipitation of cross-linked lysates. Addition of increasing amounts of genomic input DNA ensured PCRs were performed in the linear range. Results are representative of 2 independent experiments.