Cleavage of full-length STAT5α in FDC-P1(M) cells by a nuclear-associated protease from FDC-P1 cells. Unstimulated (−) or IL-3–stimulated (+) FDC-P1 and FDC-P1(M) cells were lysed and nuclear (N) and cytoplasmic (C) extracts were prepared. Extracts from FDC-P1 and FDC-P1(M) cells were analyzed either separately (lanes 1 through 4) or mixed and incubated for 15 minutes on ice (lanes 5 through 10). STAT5 proteins were detected by Western blotting using anti-STAT5 antibodies (89p). The trace levels of STAT5α observed in lane 10 are due to the addition of high levels of STAT5α in FDC-P1(M) cytosolic extracts (see Fig 2). This could be eliminated by raising the incubation temperature for 2 minutes at 37°C (data not shown).