Changes in Mcl-1 protein stability induced by TNFα. (A) Neutrophils were incubated in the presence of the protein synthesis inhibitor cycloheximide (CHX) at 10 μg/mL, in the presence or absence of TNFα (10 ng/mL) or GM-CSF (50 U/mL). Protein lysates were prepared after 2-hour (▩) and 5-hour (■) incubation, and Mcl-1 levels were detected by Western blotting. Top panel shows a representative blot of Mcl-1 protein and GAPDH protein as a loading control (after 5-hour incubation), while the bottom panel shows mean data (± SD) of relative Mcl-1 levels (taking the signal at time zero as 100%) of 3 separate experiments. (B) Neutrophils were incubated in the presence of cycloheximide alone or with TNFα, and at 1-hour intervals, protein lysates were prepared for analysis of Mcl-1 levels by Western blotting. Inset shows representative Western blots (plus GAPDH levels as loading control), while graph shows mean values of repeat experiments (± SD, n = 5).