Phospho-affinity purification of nuclear proteins and functional examination by supershift analysis. (A) Western blot analysis of Sp1 expression and phosphorylation in parental and antifolate-resistant cell lines. Nuclear proteins from parental and antifolate-resistant sublines were purified by phospho-affinity chromatography, thereby revealing phosphorylated and unphosphorylated fractions. These proteins were then resolved by polyacrylamide gels containing SDS, transferred to a cellulose nitrate Protran membrane, and reacted with antibodies against human Sp1 as detailed in “Materials and methods.” (B, C) Antibody-mediated supershift analysis. Nuclear proteins, 6 μg of untreated extracts (B) and 4 μg of phospho-affinity-purified extracts (C), from parental and a representative antifolate-resistant cell line, EDX^R0.03, with loss of binding to the GC-box element, were first incubated with antibodies (4 μg) against Sp1 and/or Sp3 for 1 hour at 4°C. Then, binding to [³²P]-labeled GC-box oligonucleotide was determined by a Phosphorimager. The supershifted antibody-nuclear protein(s)-oligonucleotide complexes are denoted on the left by a, b, and c.