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Figure 3 | Reproductive Biology and Endocrinology

Figure 3

From: Balance between matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the cervical mucus plug estimated by determination of free non-complexed TIMP

Figure 3

Functional balance as illustrated by the TIMP activity assay. Panel (A) shows the standards as follows: lane 1: α2M (0.5 μg); lane 2: active MMP-2 (25 pg); lane 3: α2M + MMP-2; lane 4: MMP-2 + TIMP-2 + α2M. (B): TIMP activity assay applied to four different CMPs: lane 1: MMP-2 standard (10 pg), lane 2: MMP-2 + α2M. Lanes 3, 5, 7 and 9: CMP (2 μl) + MMP-2 (10 pg). Lanes 4, 6, 8 and 10: CMP + MMP-2 + α2M. MMP-2 (64 kDa) lysis bands suggest that an inhibitor in the sample prevented α2M-MMP-2 capture. (C): Titration of one inhibitor-positive plug. Lane 1–4: controls. Lanes 5–10: MMP-2 (64 kDa) lysis activity declines gradually with declining CMP concentration (2, 1, 0.5, 0.25, 0.125 and 0.0625 μl). (D): MMP-1EA-TIMP capture experiment. Lane 1–4: controls. Lane 5, MMP-1EA capture control: initial incubation of MMP-1EA (2 ng) and TIMP-1 (50 pg) followed by simultaneous addition of MMP-2 (10 pg) and a 2 M (0.5 μg); MMP-1EA forms a complex with TIMP-1 and allows α2M-MMP-2 capture resulting in disappearance of MMP-2 (64 kDa) lysis band. In lane 6–9, the MMP-1EA-TIMP capture experiment is applied to a CMP. Lane 6: CMP (2 μl); lane 7: CMP + MMP-2 (10 pg); lane 8: CMP + MMP-2 + α2M (0.5 μg); lane 9: initially CMP and MMP-1EA (2 ng) followed by MMP-2 and α2M. MMP-2 (64 kDa), proMMP-2 (72 kDa), proMMP-9 (92 kDa), MMP-9-NGAL complex (125 kDa), MMP-9 dimer (184 kDa) and the α2M-MMP-2 complexes are indicated. The analysis was performed on a 7.5% gel.

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