Figure 4
A Western blot analysis of nuclear extracts from Cos7 cells transfected with wild-type and HNF-1α variants. The 60-mm dishes of confluent cells were transfected with 10 µg of expression vectors using a modified calcium phosphate precipitation procedure (16). The 10 µL of nuclear extracts were subjected to 12% SDS-PAGE, followed by Western blotting analysis using the polyclonal anti-HNF-1α antiserum (C-19; Santa Cruz). mock, Cos7 cells transfected with no expression vector. (B) EMSA analysis of HNF-1α DNA binding activity. The 1 µL of nuclear extracts from Cos7 cells transfected with the expression vectors shown in (A) were incubated with 0.42 nmol/L 32P-labeled 1A or 4AP2 probes, as indicated. Lane 0, free probe; lane 1, band shift; lane 2, competition using a 25-fold excess of cold probe; lane 3, supershift using the polyclonal anti-HNF-1α antibody C-19. A representative experiment is shown.