KL and antigen interact synergistically in the expression and phosphorylation of AP-1–related transcription factors. Deprived and nondeprived mast cell cultures were incubated overnight with antigen-specific IgE as described for previous figures. For panels A and B, deprived and nondeprived cultures were stimulated for 1 hour with 100 ng/mL KL, antigen (Ag), or both stimulants as indicated. For panel C, 100 nM wortmannin or 10 μM Ro31-7549 was added to nondeprived cells 10 minutes before addition of antigen and the reaction was terminated 1 hour later. Immunoblots were probed with antibodies against c-Jun, ATF-2, or c-Fos proteins or the activated phosphorylated forms of these proteins (designated with the prefix “p-”). For panels D-F, nondeprived cultures were stimulated with 100 ng/mL antigen for the time periods indicated. The increase in amount of c-Jun (D) and levels phosphorylated c-Jun (E) as determined by densitometric scans are shown for a typical experiment. For panel F, the data were calculated in terms of specific activity (ie, phosphorylated c-Jun/total c-Jun). All data are representative of results from at least 3 sets of experiments.