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Fig. 4. ERK1/2 is activated by CTGF via integrin vβ3 and confers enhanced migratory ability. (A) Effect of CTGF on the activation of Akt and MAPKs. Cells were serum-starved for 24 hours; activated level of Akt and MAPKs were measured by western blotting with phosphorylation-specific antibodies. (B) The role of P-ERK1/2 in CTGF-mediated cellular migration. Migration assays were performed as described above, MCF-7/neo and MCF-7/CTGF cells were treated with dimethylsulfoxide or PD98059 (20 µM), whereas cells were seeded on the upper chambers (lower figure). Cell lysates were simultaneously analyzed by western blotting with antibodies against P-ERK1/2 and ERK1 (upper panel). (C) MDA231/neo and MDA231/AS cells were transiently transfected active MEK1, 48 hours after transfection, cells were trypsinized and assayed using a Boyden chamber (lower figure). Simultaneously, cell lysates were analyzed by western blotting with antibodies against P-ERK1/2 and ERK1/2 (upper panel). (D) Effect of rCTGF on ERK1/2 activation in MCF-7 cells. Wild-type MCF-7 cells (5x10⁵) were seeded in 6-cm dishes, serum-starved for 24 hours and then treated with 50 ng/ml rCTGF for 20 minutes to 24 hours. P-ERK1/2 and ERK1 levels were detected by western blotting. (E) Wild-type MCF-7 cells (5x10⁵) were seeded in 6-cm dishes. Cells were serum-starved, pretreated with 5 ug/ml IgG or integrin-vβ3-blocking antibodies for 24 hours and then treated with 50 ng/ml rCTGF for 10 minutes. P-ERK1/2 and ERK1 were detected by western blotting. (F) p-ERK1/2 and ERK1 expression in MCF-7/neo and MCF-7/CTGF cells treated with integrin-vβ3-blocking antibody. Cells were treated with 5 ug/ml IgG or integrin-vβ3-blocking antibodies for 24 hours and P-ERK1/2 and ERK1 levels was measured by Western blotting. (G) Subcellular localization of CTGF-activated ERK1/2 analyzed by western blotting. Cells were serum-starved for 24 hours, and the nuclear and cytosolic fractions were isolated (see Materials and Methods); P-ERK1/2 and ERK1 were detected by western blotting. (H) Subcellular localization of CTGF-induced P-ERK1/2 was analyzed by immunofluorescence staining. Cells were fixed in 3.7% paraformaldehyde and co-stained with anti-P-ERK1/2 antibody and DAPI. Arrows indicate increased expression of CTGF-induced P-ERK1/2 in nuclei of breast cancer cells.

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