PU.1 protein is detectable in normal neutrophils but not PU.1-deficient neutrophils. (A) Total protein prepared from cultured neutrophils was separated on a 10% SDS-polyacrylamide gel, blotted, and probed with an antibody directed against the C-terminus of PU.1 (Santa Cruz). Note PU.1 signal around 40 kD in 50 μg of extract from the B-cell line A20-2J (lane 2) and normal neutrophils (lane 3), but not in lanes 4 through 7 containing 50 μg (lanes 4 and 6), 100 μg (lane 5), and 250 μg (lane 7) from PU.1-deficient neutrophil cell lines (#503 and #897) derived from two different PU.1-null mice. Although lane 4 containing #503 did not react with the antiactin antibody, lanes 5 and 7 (containing a twofold and fivefold excess of the same extract) clearly reflect an excess of protein present and show no PU.1 signal. Lane 1 contains the T-cell line BW.5147.7 that does not express PU.1. (B) Nuclear protein, 2.5 to 12.5 μg, prepared from cultured cells was separated on a 12% SDS-polyacrylamide gel, blotted, and probed with a polyclonal anti–PU.1-GST fusion protein antibody. No signal was detectable in PU.1-deficient cell lines in lanes 2 and 3 even at a fivefold excess of nuclear protein compared with normal neutrophils in lane 4. Lane 1 contains the T-cell line EL-4 which does not express PU.1.