Na-Sal regulates the expression of MCL-1. (A) Na-Sal does not effect GM-CSF–induced tyrosine phosphorylation of p42/p44 MAPK. TF-1 cells were deprived of GM-CSF for 12 hours and were either left untreated (lane 1) or stimulated with GM-CSF for 10 minutes (lanes 2 through 4). Some cells were preincubated with 5 mmol/L Na-Sal for 10 minutes (lane 3) or 60 minutes (lane 4) before being stimulated with GM-CSF. Cell lysates were analyzed with a phospho-specific MAPK antibody (New England Biolabs, Beverly, MA). (B) The expression of BCL-2, Bax, and BCL-X_L is not altered by Na-Sal treatment. TF-1 cells were treated with increasing concentrations of Na-Sal (0.5, 1, 5, and 10 mmol/L) for 5 hours and the presence of BCL-2, BCL-X_L, and Bax was analyzed by Western blotting. (C) Time-dependent and (D) dose-dependent inhibition of MCL-1 expression after treatment with Na-Sal. Cells were treated with 5 mmol/L Na-Sal for the indicated times (C) or were treated with the indicated concentrations of Na-Sal for 5 hours (D). The level of MCL-1 protein was determined by Western blotting. (E and F) Densitometric scanning of (C) and (D), respectively. Scanning was performed using the Ambis nonradioactive imaging system (Ambis, Inc, San Diego, CA).