HLTF is present in several major DNA-protein complexes formed on the PCS.
(A) Western blot of the K562 nuclear extract with HLTF antibody A and B shows a prominent band of 115 kd. A minor faint band below may be the degradation product. K562 nuclear extract (50 μg) was resolved on a 12% SDS-PAGE. The HLTF antibodies are indicated on the top, and the molecular weight markers are indicated at the left-hand side. (B) EMSA with 32P-labeled PCS oligonucleotide (oligo 1) and K562 nuclear extracts in the presence (+) and absence (−) of HLTF antibodies A and B. Normal rabbit serum and antibodies against p18 Maf and Sin3A served as control. In a standard 20-μL EMSA reaction mixture, 2 μL each of the antibody mentioned at the top of the panel and 5 μg nuclear extracts were preincubated for 10 minutes before the addition of the 32P-labeled DNA probe. The binding mix was incubated for an additional 10 minutes at room temperature after the addition of the DNA probe and analyzed on a 5% polyacrylamide gel.
