GM-CSF–induced tyrosine phosphorylation of STAT5B in BM-derived macrophages from WT, HZ, and STAT5A null mice. (A) BM-derived macrophages from WT (lanes 1 and 2), HZ (lanes 3 and 4), and null (lanes 5 and 6) mice were incubated with either medium (lanes 1, 3, and 5) or 10 ng/mL of GM-CSF (lanes 2, 4, and 6) for 15 minutes at 37°C and then solubilized in a buffer containing 1% Triton X-100. STAT5B was immunoprecipitated from the lysates with anti-STAT5B, and the immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting as described in Materials and Methods. The membrane was probed with the antiphosphotyrosine MoAbs PY20 and 4G10, and developed by enhanced chemiluminescence. STAT5B^P denotes tyrosine phosphorylated STAT5B. (B) The amount of protein loaded within each lane of the gel was determined by reprobing the immunoblot with the immunoprecipitating anti-STAT5B antibody and developing the blot with nitroblue tetrazolium chemistry.