IGFBP-3 binds to and inactivates Erk. (A,C-D) H1299 cells were pretreated with rBP-3 for 3 hours (A) or transiently transfected with pEV or pBP-3 (C), or with pEV-Flag or pBP-3-Flag (D) for 2 days. Lysates from H1299 cells were used for immunoprecipitation with rabbit anti-Erk1/2 antibody. Erk1/2, Flag, IGFBP-3, and p38α were detected by Western blotting. Whole cell lysates (WCLs) were subjected to a Western blot assay for phospho-Erk and phospho-p38 to determine specific inhibition of Erk by IGFBP-3. EV: empty vector. pBP-3-Flag: pCMV6-IGFBP-3-Flag. (B) Ni-NTA bead-bound his-tagged rBP-3 was incubated with H1299 whole cell lysate (150, 300, or 450 μg) for 5 hours before being washed and subjected to Western blot analysis for Erk1/2, p38α, and IGFBP-3. (E) H1299 cells were incubated with rBP-3 for 3 hours and stimulated with 10% FBS; samples were removed at different time points, as shown. The cell lysates were used for immunoprecipitation with rabbit anti-Erk antibody. Western blotting was used to detect IGFBP-3 and Erk from immunoprecipitation or whole cell lysates.