Effect of verteporfin photosensitization on κB binding activity in HL-60 cells.
(A) Nuclear extracts were prepared at increasing times after the light irradiation of cells incubated with different amounts of verteporfin. Equal amounts of protein were incubated with a 32P-labeled κB probe and separated in 6% acrylamide gels. Nonspecific and specific DNA-protein complexes are indicated. For competitor studies, nuclear extracts prepared from cells treated with verteporfin (50 ng/mL) and light 2 hours earlier were incubated with the32P-labeled κB probe in the presence of a 10 mol/L excess of unlabeled wild-type or mutated κB preparations. (B) Protein-κB complexes were characterized by gel supershift analysis. Nuclear extracts prepared from cells treated with verteporfin (50 ng/mL) and light 2 hours before were mixed with the 32P-labeled κB probe in the presence of antisera against p50, RelA, c-Rel, or RelB. Samples were separated in 4% polyacrylamide gels under nonreducing conditions. Arrows indicate the positions of κB complexes. (C) The status of IκBα was monitored after photosensitization of HL-60 cells with verteporfin and light. Cytoplasmic extracts were prepared at various times after light irradiation and were analyzed by Western immunoblot (WB) analysis using mouse monoclonal antibody MAD3 10B developed against amino acids 21-48 within the N-terminal region of IκBα. The corresponding nuclear extracts were analyzed by electrophoretic mobility shift assay as performed in part A.
