NaSal treatment results in release of cytochromecand Smac and caspase activation, but not the reduction of A1. (A-B) Monocyte-differentiated macrophages were treated with NaSal (20 mM) or LY294002 (50 μM) for indicated periods in panel A and for 48 hours in panel B, then cytosolic fraction of cells were used for Western blot assay using anti—cytochrome c antibody and anti-Smac antibody. (C) Macrophages were treated with NaSal (20 mM) for 24 or 48 hours. Cell lysates were collected and Western blot assays were conducted using the indicated antibodies. (D) Macrophages were infected with adenoviral vector (AdNIK, 100 moi) expressing NR-κB—inducing kinase for 24 hours as described in “Materials and methods.” Then cells were treated with NaSal at 20 mM for another 24 hours. Cells were harvested and total RNA was extracted for RT-PCR using human A1 and GAPDH-specific primers as was previously described by us.20 No indicates normal macrophages without NaSal treatment.
