FIGURE 3.
TIMP-1 interferes with MMP-9 homodimerization. A, TIMP-1, but not TIMP-2 interferes with MMP-9 dimerization in transfected COS-1 cells examined by a co-immunoprecipitation (IP) assay for conditioned medium (CM) and cell lysates (upper and middle panels). Western blotting (WB) using an aliquot of the conditioned medium and cell lysates (CL) was performed using anti-Myc, HA, TIMP-1, TIMP-2, and α/β-tubulin antibodies (lower panel, loading control). B, TIMP-1 co-precipitated with MMP-9 in both the cell lysate and the conditioned medium of transfected COS-1 cells examined by a co-immunoprecipitation assay. The reciprocal co-immunoprecipitation experiment is shown in supplemental Fig. 1E. The conditioned medium and cell lysate were examined by Western blotting using anti-TIMP-1 and α/β-tubulin antibodies for control of protein expression. C, MMP-9/PEXMMP-2/Myc, but not wild type MMP-9, co-precipitates with TIMP-2. COS-1 cells were co-transfected with a combination of cDNAs as indicated. The conditioned medium and cell lysate were examined by a co-immunoprecipitation assay. Aliquots of conditioned medium and cell lysate were examined by Western blotting using anti-TIMP-2 and MMP-9 antibody, respectively, for control of protein expression. D, TIMP-1, but not TIMP-2, interferes with MMP-9-induced cell migration. COS-1 cells transfected with corresponding cDNAs, as indicated, were subjected to a transwell migration assay. Three triplicate repeats were performed for each transfection and the experiment was repeated three times. *, p < 0.05.