PU.1 is detectable in 503 cells transduced with a PU.1-expressing retrovirus. PU.1-deficient 503 cells were transduced with retroviral vectors containing the cDNA for PU.1, G-CSF receptor, or M-CSF receptor. Cells that were positive for the selectable marker PLAP (PU.1 and G-CSF receptor) or positive for growth in M-CSF (M-CSF receptor) were isolated for analysis. (A) RT-PCR showed PU.1 mRNA in normal macrophages (MAC) and PU.1-transduced 503 cells (503-PU), but not in 503 or 503 transduced with M-CSF receptor (503-MR). Primers used were intron-crossing, and controls for DNA amplification in the absence of RT were also included and were negative (not shown). The far righthand lane contains a 100-bp DNA ladder for size determination of PCR products. (B) Whole-cell lysates were prepared and 50 μg (or 80 μg where indicated) of total cellular protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After blotting and probing with a polyclonal anti-PU.1 antibody, PU.1 protein was visible in normal mixed myeloid cells, A20-2J B cells, and 503-PU cells. As expected, nonhematopoietic HT1080 cells were negative for PU.1 expression. The lane labeled MW contains protein size standards (CruzMarker; Santa Cruz Biotechnology). MW, molecular weight.