Cells were grown and plated as described in Materials and Methods. A, Cells were pretreated for 1 h with vehicle, 20 μM PD98059, or 10 μM U0126, and then treated ± 4 nM PRL for 15 min. Cell lysates were harvested, and equal amounts of protein were analyzed by Western blot for phospho-ERK1/2, phospho-ERK5, and phospho-JNK1/2. B, Cells were cotransfected with 4XAP-1-luc, lPRLR, and β-galactosidase. After transfection, cells were pretreated for 1 h with vehicle or 10 μM U0126, and then treated ± 4 nM PRL for 6 h in the presence of inhibitor. C, D, and E, Cells were cotransfected with 4XAP-1-luc, lPRLR, β-galactosidase, and DN ERK1 and DN ERK2 or vector (C) or DN JNK1 and DN JNK2, DN ERK5, p38α KD, or vector (D) or increasing concentrations of JIP-1 (34) or vector (E). After transfection, cells were allowed to recover overnight in serum-free media and then treated ± 4 nM PRL for 6 h. All lysates were assayed for luciferase and β-galactosidase activity as described in Materials and Methods. In B, C, and D, relative activity represents the mean of the corrected luciferase activity from at least three independent experiments, represented as mean fold change relative to the vehicle-treated cells in the absence of inhibitor or dominant negative construct ± SEM. For statistical analyses, PRL- and vehicle-treated samples similarly transfected, or treated with inhibitor, were compared. Asterisks denote significant differences between vehicle- and PRL-treated cells (P < 0.03) using Student’s t test.+, Significant difference from vehicle-treated control transfection (P < 0.05) using Student’s t test. E is a representative experiment with each data point in triplicate ± SD and graphed as relative luciferase units (RLUs).