FIG. 3.
VP16 activates transcription in association with HCF-1 and CeHCF but only weakly in association with HCF-2. (A) Transcriptional activation by VP16 in tsBN67 cells grown at 33.5°C. The cells were transfected with an HCF expression construct, a VP16 expression construct, and reporter constructs, and the resulting β-globin and α-globin reporter RNAs were probed by RNase protection analysis as described in Materials and Methods. Odd-numbered samples contained no cotransfected VP16 expression vector. The samples shown here were normalized to the level of internal control α-globin transcript. The positions of the RNase-protected fragments corresponding to α-globin (α), correctly initiated β-globin (β), and read-through (RT) β-globin transcripts are indicated on the left. +, present; −, absent. (B) Quantitation of relative β-globin transcript levels. The intensity of each band corresponding to the β-globin transcript in the samples containing VP16 was measured by phosphorimager analysis. The transcript levels are shown relative to the HCF-1N1011 sample (panel A, lane 4). The results represent the average of two complete experiments; the CeHCFN395 was uncharacteristically high in the experiment not shown here (high error bars), and its apparently higher activity than CeHCFFL shown here is not a true representation of its activity. (C) Immunoblot analysis of the tsBN67 cell extracts used in the in vivo transcription assay shown in panel A. Extracts were resolved by sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane, and the HA-tagged HCF and VP16 proteins were detected with the 12CA5 anti-HA antibody. Only the VP16-containing samples are shown. The position of each relevant HCF species is indicated with a black dot to the right of each lane. A long exposure of lane 4 is shown on the right (lane 4′). Relative levels of HCF-protein synthesis are indicated above each lane. Lane 1, no ectopic HCF protein synthesized.