Fig. 4. Foxa2 inhibits En2SP binding to the HF1 and HF2 sites of MAP1B promoter. (A) Binding of En2SP and Foxa2 to the HF1 site. The HF1 probe sequence, highlighting the ATTA (square box) and Foxa2 (oval box) sites, is represented above the EMSA panels. Purified En2SP or GST-Foxa2 fusion proteins were incubated independently (lanes 2-7), or in combination (lanes 8-9). Lane 1: free HF1 probe; lanes 2-3: 25 and 50 ng of En2SP, respectively; lanes 4-5 and 6-7 show two different autoradiographic exposures of the same gel area (4-5: long exposure; 6-7: short exposure). Gels were loaded with 40 ng (lanes 4 and 6) or 120 ng (lanes 5 and 7) of GST-Foxa2. An interaction of Foxa2 with HF1 is visible in lane 5 (double arrowhead). At low concentration (lane 2: 25 ng) En2SP gives one retarded band, while at a higher one (lane 3: 50 ng) it generates two retarded bands (arrowheads). Co-incubation of GST-Foxa2 with En2SP leads to an inhibition of En2SP binding (compare lane 3 to lanes 8 and 9). (B) Binding test for En2SP and Foxa2 to the HF2 site. The probe sequence, highlighting the ATTA (square box) and Foxa2 (oval box) sites, is represented above the EMSA panel. Purified En2SP or GST-Foxa2 fusion protein were incubated independently (lane 2: 50, lane 3, 100 ng of En2SP; lane 4: 120, lane 5, 200 ng of GST-Foxa2) or together (lane 6-9) with the HF2 probe. En2SP was preincubated with HF2 for 20 minutes, before adding Foxa2. En2SP binds HF2 (arrowhead), GST-Foxa2 does not bind by itself (lanes 4,5) but dissociates En2SP from HF2 (compare lane 2 with lanes 6-7, and lane 3 with lanes 8-9).