The STAT5A/WKR, EE, and VVV mutants lack FcγRI GAS DNA-binding activity. Nuclear extracts were prepared from 293T cells transfected with JAK2 and the indicated murine STAT5A mutants and analyzed by EMSA using a ³²P-labeled FcγRI-derived GAS probe. 293T cells transfected with either parental STAT5A (5A) alone or JAK2 (JK2) alone are shown at left. STAT5-specific GAS DNA-binding activity was confirmed by incubation with anti-STAT5A antibody (5A+JK2+AB). The positions of the STAT5 GAS DNA-binding complex (lower arrowhead) and the supershifted complex (upper arrowhead) are indicated. Similar supershifts using anti-HA antibody were observed for the STAT5A/RQQ and STAT5A/▵53C complexes (data not shown).