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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. 8. — KIX and CH3 domains interdependently regulate Zac coactivation. (A) Immunoblot of PA-TU cells expressing adjusted amounts of p300 or derivatives as detected by α-Flag (10 μg of pCI.Flag-p300, 5 μg of pCI.Flag-p300ΔKIX, 3 μg of pCI.Flag-p300ΔCH3, or 1 μg of pCI.Flag-p300ΔKIX ΔCH3). (B) EMSA with DR element. PA-TU cells were transfected with Zac in the absence or presence of adjusted amounts of p300, p300ΔKIX, p300ΔCH3, or p300ΔKIXΔCH3. Zac-DNA complexes () were unaffected by preimmune-N or α-p300-N sera in the absence of p300 (lanes 1 and 2). They were indistinguishably supershifted () by α-p300-N but not by preimmune-N serum in the presence of p300, p300ΔKIX, or p300ΔCH3 (lanes 3 to 8), while p300ΔKIXΔCH3 was ineffective (lanes 9 and 10). (C and D) Zac (0.1 μg of pRK7Flag-Zac) and DR or PAL reporters (2 μg each) were cotransfected with increasing amounts of p300, p300ΔKIX, and p300ΔCH3 into PA-TU cells. Indicated amounts of DNA refer to p300 and appropriately adjusted amounts of the derivatives.

Inline graphic — KIX and CH3 domains interdependently regulate Zac coactivation. (A) Immunoblot of PA-TU cells expressing adjusted amounts of p300 or derivatives as detected by α-Flag (10 μg of pCI.Flag-p300, 5 μg of pCI.Flag-p300ΔKIX, 3 μg of pCI.Flag-p300ΔCH3, or 1 μg of pCI.Flag-p300ΔKIX ΔCH3). (B) EMSA with DR element. PA-TU cells were transfected with Zac in the absence or presence of adjusted amounts of p300, p300ΔKIX, p300ΔCH3, or p300ΔKIXΔCH3. Zac-DNA complexes () were unaffected by preimmune-N or α-p300-N sera in the absence of p300 (lanes 1 and 2). They were indistinguishably supershifted () by α-p300-N but not by preimmune-N serum in the presence of p300, p300ΔKIX, or p300ΔCH3 (lanes 3 to 8), while p300ΔKIXΔCH3 was ineffective (lanes 9 and 10). (C and D) Zac (0.1 μg of pRK7Flag-Zac) and DR or PAL reporters (2 μg each) were cotransfected with increasing amounts of p300, p300ΔKIX, and p300ΔCH3 into PA-TU cells. Indicated amounts of DNA refer to p300 and appropriately adjusted amounts of the derivatives.