Binding of the recombinant 38-kd HLTF to wild-type and mutated PCS.
A cDNA clone coding for the N-terminal 38-kd HLTF (aa 2 to aa 332) isolated by screening the λ gt11 cDNA library of K562 cells was cloned into pTrcHis2TOPO vector and expressed in E colicells. The protein extracts of uninduced and IPTG-induced bacterial cells were used in the EMSA. The wild-type and mutant versions of the PCS oligonucleotides are indicated, and their sequences are described in Figure 5. The 20-μL EMSA binding mixture contained 2 ng32P-labeled double-stranded DNA (∼100 000 cpm) in the binding buffer and 1 μL E coli protein extract. The binding reaction was started by the addition of the32P-labeled DNA probe. The reaction mixture was incubated for 10 minutes at room temperature and then analyzed on a 5% polyacrylamide gel. For gel supershift/neutralization assay with antibody A, the bacterial extract, and the antibody were incubated for 5 minutes before the addition of the 32P DNA probe. The EMSA band obtained with 38-kd HLTF is shown by an arrow. The upper EMSA band is due to a bacterial protein in the extract.
