PRL enhances IGF-I-induced IGF-IR phosphorylation and activation of downstream signaling pathways. Serum-starved MCF-7 cells were treated with vehicle, IGF-I, PRL, or IGF-I/PRL for the indicated times. The immunoblots were performed using cell lysates and the indicated antibodies. A, representative experiment. B, levels of p-IGF-IR/total IGF-IR in response to IGF-I alone, compared with IGF-I + PRL, quantified as described under “Experimental Procedures” (means ± S.D., n = 3). The asterisks denote significant differences compared with IGF-I treatment. ** p < 0.01 (two-way ANOVA, Bonferroni post-test). C and D, levels of phosphorylated ERK1/2 and AKT, respectively, compared with total kinase levels, quantified as described under “Experimental Procedures” (means ± S.D., n = 3). The asterisks denote significant differences compared with IGF-I treatment. *, p < 0.05 (paired t test).