pDP4 promoter activity is regulated by PU.1. (A) COS-7 cells were transiently transfected with pXP2-pDP4-prom (300 ng), a plasmid that expresses a luciferase gene under the transcriptional control of the proximal 0.4-kb pDP4 promoter, along with (200 ng each) pcDNA3, pcDNA3-PU.1, or pcDNA3-ICSBP, and the reference vector pRL-CMV (50 pg). The graph shows a representative experiment of 3 independent assays ± standard deviation. (B) WEHI-3 cells, which express endogenous pDP4, were transiently transfected with (1.9 μg each) either a promoterless pXP2, pXP2-pDP4-prom, or pXP2-pDP4-prom(mut.) carrying a mutated (TTCC to GCGA) PU.1-binding site, along with the control plasmid pRL-TK (100 ng). The graph shows a representative experiment of 2 independent assays. (C) Real-time RT-PCR of pDP4 transcripts from bone marrow (BM), 503, or 503/PU.1 cells. The results were normalized to 18S rRNA values. (D) Western blot of total cell extracts (60 μg) from spleen, bone marrow, the 503 PU.1^-/- line, and the 503/PU.1 line showing the absence of pDP4 protein expression in 503 cells before the retroviral restoration with PU.1. The upper panel was immunoblotted with the pDP4 antiserum while the lower panel demonstrates equal protein loading as judged by immunoblotting with an anti-actin antibody.