Figure 2
Control CAT assays to examine repression of the TK-CAT promoter-reporter in U2OS cells by p68/p72. The amounts of DNA transfected in each assay are indicated below and in all cases the % conversion of 14C-labelled chloramphenicol to acetylated forms is shown as an average of three independent experiments. a) Effect of untagged p68/p72 on TK-CAT transcription. 7.5 Ī¼g of control pcDNA3 vector, pcDNA3-p68 (p68) or pcDNA3-p72 (p72) were co-transfected with 2.5 Ī¼g of TK-CAT. b) Effect of GAL4-tagged p68 and p72 on transcriptional activity of a TK-CAT promoter-reporter which incorporated a 1.6 kb DNA 'spacer' between the GAL4 binding sites and the promoter (TK-S-CAT). 1 Ī¼g of pcDNA3-GAL4 (pcG4) or GAL4-tagged p68/p72 (p68G4/p72G4) were co-transfected with 5 Ī¼g of TK-S-CAT. The amount of TK-S-CAT had previously been titrated to achieve an appropriate baseline level of CAT activity. c) Titre of repression of TK-CAT activity by GAL4-tagged p68/p72. 2.5 Ī¼g of TK-CAT were co-transfected with different amounts of pcG4 vector, p68G4 and p72G4 as indicated. d) Effect of p300 and CBP on repression of TK-CAT transcription by GAL4-tagged p68/p72. 2.5 Ī¼g of TK-CAT were co-transfected with 1 Ī¼g of pcG4 vector, p68G4 or p72G4 together with 6.5 Ī¼g of either bluescript (as control) or p300/CBP.