Translocalization and colocalization of rBP-3 with Erk in H1299 cells. (A) H1299 cells on cover slips were treated with rhIGFBP-3 (10 μg/mL) and fixed at 0, 10, 30, and 60 minutes after being washed 3 times in PBS. Nuclei (blue) were stained with Hoechst 33342 (1 μg/mL). Erk1/2 (green) was stained with mouse anti-Erk antibody and secondary antibodies conjugated with Alexa Fluor 488. IGFBP-3 (red) was stained with rabbit anti-IGFBP-3 antibody and secondary antibodies conjugated with Alexa Fluor 568. For the negative control, cells treated with rhIGFBP-3 for 60 minutes were stained with secondary but not primary antibodies. Neg Con: negative control. Colocalization map: schematic plot of colocalization; white dots represent the colocalization of Erk and IGFBP-3. The empty arrowhead indicates IGFBP-3 accumulation near the cell membrane. (B) Schematic model of IGFBP-3's angiogenesis inhibition. IGFBP-3 inhibits tumor angiogenesis by IGF-dependent and -independent mechanisms. In the IGF-independent mechanism, IGFBP-3 directly binds to and inactivates Erk1/2, prevents Elk-1 activation and binding between activated Elk-1 and Egr-1 promoter, and inhibits expression of Egr-1 and its target genes, including bFGF and PDGF, resulting in suppression of angiogenesis and tumor growth.