Fig. 2. Binding of Foxa2 to fragment C of MAP1B promoter. (A) Purified GST (lanes 2-4; 20, 40 or 80 ng) or GST-Foxa2 fusion proteins (lanes 5-7; 20, 40 or 80 ng; lane 9: 20 ng) were incubated with radioactive MAP1B promoter fragment C (lanes 1-7) or fragments C plus E (lanes 8-9). Migration of the free probe is indicated on the left. GST-Foxa2 binds to fragment C, but not E, in a dose-dependent manner; GST alone shows no binding activity. (B) Binding of 1 µg nuclear extracts from P0 mice cerebellum to fragments C, D, E and C + E. Migration of free probes is indicated on the left. Both fragments C and D are retarded by the cerebellar nuclear extracts. The complex bound to fragment C contains Foxa2, as demonstrated by the supershift induced by the addition of the anti-Foxa2 antibody (4C7 Ab). No supershift is observed when the 4C7 antibody is used in the absence of cerebellum nuclear extracts. No supershift is observed when another — unrelated — monoclonal antibody (anti-myc, 9E10) is used instead of the 4C7 antibody (CTRL Ab, right panel).