p42/p44 MAPK activity is prolonged when protein synthesis is blocked by CHX and unaffected when the proteasome function is blocked by LLnL. Activity of immunoprecipitated p42/p44 MAPK was analyzed as described in Materials and Methods. MAPK activity is expressed as fold stimulations over basal activity. (A) Lane 1, CCL39 cells were left untreated; lanes 2 and 3, cells were treated with CHX and LLnL, respectively, for 3 h; lanes 4–6, cells were stimulated 7 min with 10% FCS; lanes 7–9, cells were stimulated for 1 h with 10% FCS; lanes 10–12, cells were stimulated for 3 h with 10% FCS. CHX (30 μg/ml) was added 15 min before FCS in lanes 5, 8, and 11 (black bars). LLnL (30 μM) was added 15 min before FCS in lanes 6, 9, and 12 (hatched bars). (B) (Separate experiment) Lane 1, CCL39 cells were left untreated; lanes 2 and 3, cells were stimulated for 1 h with 10% FCS; lanes 4 and 5, cells were stimulated 1 h with 100 ng/ml TPA; lanes 6 and 7, cells were stimulated for 3 h with 10% FCS; lanes 8 and 9, cells were stimulated for 3 h with 100 ng/ml TPA; lanes 10 and 11, cells were stimulated for 12 h with 10% FCS; lanes 12 and 13, cells were stimulated for 12 h with 100 ng/ ml TPA. LLnL (30 μM) was added 15 min before FCS or TPA in lanes 3, 5, 7, 9, 11, and 13 (hatched bars).