FIG. 2.

Characterization of Oct-1 protein levels in mutant cells. (A) DNA binding activity is decreased in both heterozygous and homozygous MEF nuclear extracts. Lanes contained 5 μg of nuclear extract. HeLa extract was included as a positive control (lane 2). Lanes 3 and 5 contained wild-type extract, lane 4 had extract from heterozygous MEFs, and, lane 6 had extract from Oct-1-deficient MEFs. Oct-1/DNA complex is indicated by an asterisk. (B) Oct-1 DNA binding activity is dramatically reduced in mutant nuclear extract. EMSA of nuclear extract derived from wild-type (lanes 3 to 5, 11) and Oct-1-deficient immortalized cell lines (lanes 6 to 8, 12). Lanes contained 10 μg of total protein. **, supershifted complex formed upon addition of anti-Oct-1 antibody (lanes 4 and 7). Reactions in lanes 1 to 8 were incubated with an octamer oligonucleotide. and reactions in lanes 9 to 12 were incubated with a mutant. (C and D) A 1-s exposure and 2-min exposure, respectively, of a Western blot with a combination of three anti-Oct-1 antibodies (Materials and Methods). The load consisted of 2.5 μg of nuclear extract (lanes 1 and 2), representing 1% of the input sample. Truncated polypeptides are indicated by an asterisk (lane 8). Nuclear extract load (L[NE]), flowthrough (FT), wash (0.2 M W), and elution (1.0 M).