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. Author manuscript; available in PMC: 2007 Sep 19.
Published in final edited form as: J Biol Chem. 2007 May 18;282(30):21998–22010. doi: 10.1074/jbc.M701737200

FIGURE 7. MT6-MMP is not detected in complex with TIMPs.

FIGURE 7

A–D, MT6-MMP and GPI-MT1-MMP were expressed in BS-C-1 cells by infection-transfection as described under “Experimental Procedures.” Six hours post-infection-transfection, the cells received 20 nm of purified recombinant TIMP-1 or TIMP-2 in serum-free DMEM followed by overnight incubation. The media were removed, and the cells were treated with (+) or without (−) PI-PLC. The supernatants were collected, concentrated, and resolved by reducing 12% SDS-PAGE followed by immunoblot analyses. The blots were probed successively with mAb101 to TIMP-2 (A), mAb LEM-2/15 to MT1-MMP (B), mAb to TIMP-1 (C), and pAb Ab39031 to the hinge of MT6-MMP (D). The asterisks in D show nonspecific bands. Recombinant TIMP-2 (A, lane 5) and TIMP-1 (C, lane 5) (10 ng each/lane) were run as a control. E, MT6-HCT and EV-HCT cells were cultured in the presence of 20 nm of TIMP-1, TIMP-2, TIMP-3, or TIMP-4 in complete media. The cells were then surface-biotinylated as described under “Experimental Procedures.” The biotinylated cells were then treated with PI-PLC, and the supernatants were collected and subjected to streptavidin beads pulldowns. The bound proteins were eluted with reducing Laemmli SDS-sample buffer and resolved by 12% SDS-PAGE followed by immunoblot analysis. The blots were probed with antibodies to MT6-MMP or to TIMPs. Only the blot probed with MT6-MMP antibody (mAb1142) is shown. The TIMP blots showed no signals. Arrows in A and C indicate the recombinant TIMP-2 (∼22 kDa) and TIMP-1 (∼30 kDa), respectively.