c-Myb binds to GATA-3 through the adaptor protein Menin. (A) Immunoprecipitation (IP) was performed with human primary CD4⁺ cells stimulated for 5 days under Th2 cell-promoting conditions. Anti-Menin, anti-c-Myb, and anti-GATA-3 antibodies and mouse IgG (mIgG) were used for the IPs. The Western blot was performed with anti-Menin antibody. The blot is representative of 2 independent experiments. (B-C) IPs were carried out with anti-Flag antibodies. Proteins used were synthesized by in vitro translation, except for the top panel of Figure 5C, where CD4⁺ T-cell lysate stimulated for 5 days under Th2 cell-promoting conditions was also used. The precipitated proteins were detected with Menin (B), GATA-3 (C, top and middle panels), or c-Myb antibodies (C, bottom panel). F indicates Flag-tag; and F-325, truncated c-Myb protein without its negative regulatory domain (1-325 amino acids of c-Myb). The blots are representative of at least 2 independent experiments.