Time-course analysis of ERK1/2 and p38/MAPK phosphorylation in response to TRAIL. RAW264.7 cells were made quiescent by reduction of serum to 0.5% in culture medium overnight, before stimulation with TRAIL and RANKL plus M-CSF, used alone or in combination. Equal amounts of cell lysates, harvested at the indicated time points, were analyzed for ERK1/2 (A) and p38/MAPK (B) phosphorylation by Western blot analyses using Abs specific for the native form of the kinases and for residues that are phosphorylated (P-) in each kinase upon activation. Protein bands were quantified by densitometry and levels of P-ERK1/2 (A) and P-p38 (B) were calculated for each time point, after normalization to ERK1/2 or p38, respectively, in the same sample. Unstimulated basal expression was set as unity. Error bars indicate SD. Results are representative of 3 separate experiments.