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. 1999 Oct;19(10):6509–6522. doi: 10.1128/mcb.19.10.6509

FIG. 2.

FIG. 2

The F domain of HNF4α1 inhibits transcriptional enhancement by coactivators. (A) Diagram of promoter region of reporter construct pZLHIVAI-4. HIV.LTR, human immunodeficiency virus long terminal repeat. (B to D) Transient cotransfections into 293T cells were performed as described in Materials and Methods with 2 μg of reporter construct, 0.5 μg of various HNF4α1 expression vectors as described in Fig. 1, and various amounts of GRIP1 (pSG5.GRIP1 full length), p300 (CMV.HA.p300 partial), or CBP (pRC.RSV.HA.CBP full length) expression vectors as indicated. Shown is the average fold induction of the relative light units normalized to a β-Gal control (0.5 to 1.0 μg of RSV.βgal) from one of several experiments. Error bars indicate the range of triplicate samples. Panel C and minus in panel D, no added coactivators. (D) One microgram GRIP1 and 5 μg of CBP expression vectors were used. Note the difference in scale of the y axes in panels C and D. (E) Electrophoretic mobility shift analysis of crude nuclear extracts from COS-7 cells transiently transfected with the various HNF4α expression vectors as indicated (HNF4α1 and HNF4α2, 25 μg of expression vector per 1.6 × 106 cells; N45C455, N1C374, and N45C374, 50 μg per 3.4 × 106 cells). DNA was introduced into the cells by standard calcium phosphate procedure, and the cells were harvested 40 h after glycerol shock. 32P-labeled APF1 oligonucleotide (0.5 ng per 7.5-μl reaction) was incubated with the protein extract (0.5 μg) in the presence of nonspecific DNA (0.5 μg of dI-dC, 0.5 μg of sonicated salmon sperm DNA) for 20 min at room temperature before the addition of antiserum (0.5 μl) as indicated. The incubation was continued for another 20 min before electrophoresis on a 6% native polyacrylamide gel. Details of procedures have been described previously (44, 78). Lanes: −, no antiserum added; PI, preimmune antiserum; α445, antiserum to the C terminus of rat HNF4α1 (80); αN1.14, antiserum raised in rabbit to a synthetic peptide corresponding to the first 14 aa of the N terminus of rat HNF4α1.