Figure 3

Identification and characterization of a novel protein/DNA complex forming specifically at the mutation identified in the patient. A) Protein binding to c-fos promoter and mapping of nucleotides necessary for complex formation. Radiolabeled c-fos-wt or mutated pc-fos-c.–439 T→A DNA fragments were subjected to DNaseI protection assay with increasing amounts (◄: 4 μg, 8 μg, 20 μg, 40 μg) of liver cells (HepG2) nuclear protein extracts and digested with varying DNaseI concentrations (◄: 0.11U, 0.33U to 1.0U). A protein/DNA interaction does solely occur with c-fos–c.-439T>A. The sequence of protected areas P1 (nt −413 to −403 (CCCAGCCGCGG) P2 (nt −441 to −428 (CAA TCTGCGCCGTT) and P3 (nt −458 to −455 (GTGC) indicated the mutation being located in P2. A typical result from 5 experiments is shown (lane GA: purine sequence ladder). B) EMSA with nuclear protein extracts from liver cells (HepG2) using c-fos-wt (−451 to −430) or mutated pc-fos-c.–439 T→A as probe. The specific complex is indicated by an arrow. Mutation specific complex formation was tested by cross competition with 100-fold excess of non radiolabeled fragments of either c-fos-wt or pc-fos-c.–439 T→A (lane F. free probe, 1: no competition; competition: 2: 50x, 3: 100x specific competitor, 4: 100x cross competition) C) Size fractionation of EMSA protein band on denaturing SDS PAGE. All resulting protein bands were subjected to mass spectrometry. Acquired data from each individual spot were used to search a human sub-set of Swiss-Prot (Sprot_2011; 20249 protein entries) for protein identification.