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. 2006 Jul;26(14):5544–5557. doi: 10.1128/MCB.02270-05

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Copyright © 2006, American Society for Microbiology

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FIG. 5. — Zac zinc fingers and p300 form a DNA-bound complex. (A) Scheme of p300. Fragments used for immunization are underlined; numbers denote amino acids. The corresponding antibodies are labeled α-p300-N and α-p300-C. SID, steroid receptor interacting domain; BD, bromodomain. (B) EMSA. PA-TU cells were transfected with Zac (0.2 μg of pRK7Flag-Zac) in the absence or presence of p300-HA (1.0 μg of pCMV-p300-HA). Zac-DNA complexes on the DR element () are supershifted by anti-Flag antibodies (). Anti-p300 antibodies (α-p300-N and α-p300-C) were ineffective in the absence of ectopic p300 (lanes 3 and 4) but strongly supershifted Zac-DNA complexes () upon p300 cotransfection (lanes 7 and 8). Preimmune sera were ineffective (lanes 9 and 10). (C) EMSA. Adjusted amounts of Zac (0.2 μg of pRK7Flag-Zac) and ZacΔC1 (0.04 μg of pRK7Flag-ZacΔC1) were transfected in PA-TU cells and tested as described above.

Inline graphic — Zac zinc fingers and p300 form a DNA-bound complex. (A) Scheme of p300. Fragments used for immunization are underlined; numbers denote amino acids. The corresponding antibodies are labeled α-p300-N and α-p300-C. SID, steroid receptor interacting domain; BD, bromodomain. (B) EMSA. PA-TU cells were transfected with Zac (0.2 μg of pRK7Flag-Zac) in the absence or presence of p300-HA (1.0 μg of pCMV-p300-HA). Zac-DNA complexes on the DR element () are supershifted by anti-Flag antibodies (). Anti-p300 antibodies (α-p300-N and α-p300-C) were ineffective in the absence of ectopic p300 (lanes 3 and 4) but strongly supershifted Zac-DNA complexes () upon p300 cotransfection (lanes 7 and 8). Preimmune sera were ineffective (lanes 9 and 10). (C) EMSA. Adjusted amounts of Zac (0.2 μg of pRK7Flag-Zac) and ZacΔC1 (0.04 μg of pRK7Flag-ZacΔC1) were transfected in PA-TU cells and tested as described above.