Both activated and nonactivated forms of STAT5α are cleaved by the protease in FDC-P1 nuclear extracts. The different forms of STAT5α were isolated from either stimulated FDC-P1(M) nuclear extracts (N) or unstimulated cytosol extracts (C) by immunoprecipitation using antisera that recognizes the carboxyl terminus of STAT5b (C-17). The Sepharose pellet was dissolved in nuclear buffer and aliquots were either mixed with 1/10 volume of nuclear extract from unstimulated FDC-P1 cells or nuclear extract buffer. Extracts were incubated on ice for 15 minutes and then transferred to 37°C for 2 minutes before freezing. STAT5 proteins were detected by Western blotting using anti-STAT5 antibodies (89p) or anti–P-Ty (4G10).