Dephosphorylation of nuclear proteins using calf intestinal alkaline phosphatase. (A) Restoration of promoter element binding after treatment with calf intestinal alkaline phosphatase in antifolate-resistant cell lines lacking GC-box binding. Drug-resistant cell lines with decreased (or loss of) binding to the cis-acting element, due to specific modifications of specific transcription factors, were treated with alkaline phosphatase. The binding to [³²P]-labeled GC-box was then determined. (B, C) Antibody-mediated supershift analysis. Nuclear proteins, 6 μg of untreated extracts (B) and 3 μg of alkaline phosphatase-treated 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. Binding to [³²P]-labeled GC-box oligonucleotide was then determined by a Phosphorimager. The supershifted antibody-nuclear protein(s)-oligonucleotide complexes are denoted on the left by a, b, and c.