Direct binding of PU.1 to cis-enhancing sequences in the CD80 and CD86 promoters. EMSA with each probe and in vitro–translated PU.1 protein. Common Ets-motif (GGAA) and PU.1-recognizable sequence (AGAA) in the probes are boxed (top). PU/IRF probe is bound with PU.1/IRF4 or IRF8 heterodimer via overlapping Ets (GGAA; boxed) and IRF (AANNGAAA; underlined) sequence.26 Protein, in vitro transcription/translation reacted mixture using empty vector pCR3.1 (E), pCR-PU.1 (PU.1, PU, or P), pCR-IRF (4), pCR-IRF8 (8), or nuclear extract prepared from PU.1-overexpressing RAW264.7 (nuclear). antibody, anti–PU.1 goat IgG antibody (PU), anti–IRF4 goat IgG antibody (4), anti–IRF8 goat IgG antibody (8), or control goat IgG (Ig). CD80-1 M1-5, CD86-1B M1-3, and CD86-2 M1 and M2 (F) are competitive oligonucleotides with mutant sequences at each indicated site (B). WT is each self competitor with a wild-type sequence (F).
