Cell-specific protein-DNA complex associated with a strong positive regulatory region (−115 to −89) of the β₃ regulatory unit. (A) Location of the probes spanning a portion of the β₃ regulatory unit that has cell-specific and potent positive regulatory function. In (B) and (C), cold specific and irrelevant oligonucleotide competitors (Table 1) were used in a 50-fold molar excess. Prefixes: c, unlabeled (cold) oligonucleotide; m, mutant oligonucleotide; cm, cold mutant oligonucleotide. (B) EMSA using³²P-labeled MSPw oligonucleotide probe incubated with the indicated nuclear extracts. Nuclear extracts used in lane 5 were from K562 cells treated with PMA, which usually, but inconsistently, showed the P1 complex. The megakaryocytic cell-specific band is indicated as P1. The intensity of the P1 signal from PMA-treated K562 cells did not correlate with β₃ RNA (see Fig 1B), suggesting that the −115 to −89 sequence is not the sole contributor to transcription. Irr., irrelevant DNA competitor (Table 1). (C) EMSA using³²P-labeled Pw, mPu1, or mPu2 oligonucleotide probes incubated with the indicated nuclear extracts. The megakaryocytic cell-specific band is again shown as P1, because it comigrates with the MSPw complex (not shown). Radiolabeled probes are Pw in lanes 1 through 10, mPw1 in lanes 11 and 13, and mPw2 in lanes 12 and 14. (*)³²P-labeled DNA probe. (D) EMSA using³²P-labeled wild-type (MS4) and mutant (mMS4) oligonucleotide probes and K562 nuclear extracts. cMS4 is unlabeled MS4. Irr., irrelevant DNA competitor.