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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 4. Cell surface MT6-MMP is displayed as an ∼120-kDa species.

FIGURE 4

A, immunoblot analyses of lysates of pooled clones of EV-HT (lanes 1 and 5), MT6-HT (lanes 2 and 6), EV-HCT (lanes 3 and 7), and MT6-HCT (lanes 4 and 8). Cells were lysed in cold lysis buffer supplemented with 20 mm NEM and mixed with Laemmli SDS-sample buffer with (+) or without (−) β-ME. The lysates (10 μg/lane) were resolved by 7.5% SDS-PAGE followed by immunoblot analysis with mAb1142. B, crude plasma membrane fraction isolated from MT6-HCT cells was treated with PI-PLC (5 units per 5 mg of total protein in 1 ml of TBS) and centrifuged, and the supernatant (lanes 3 and 6,20 μg/lane) and pellet (lanes 2 and 5, 20 μg/lane) were collected. Lanes 1 and 4 show the input plasma membrane fraction (2 μg/lane) before PI-PLC treatment. The fractions were mixed with Laemmli SDS-sample buffer with (+) or without (−) β-ME and resolved by 7.5% SDS-PAGE followed by immunoblot analysis with pAb Ab39031 to the hinge region of MT6-MMP. C, MT6-HCT cells in 6-well plates were treated with 0.5 ml of PI-PLC (0.3 units/well) in PBS for 30 min on ice. The supernatant was concentrated and mixed with Laemmli SDS-sample buffer with (+) or without (−) β-ME and resolved by 7.5% SDS-PAGE followed by immunoblot analysis with mAb1142. D and E, EV-HCT (lanes 1 and 2) and MT6-HCT tumors (lanes 3 and 4) were homogenized, and extracts were immunoprecipitated with either anti-MT6-MMP hinge pAb Ab39031 (D and E) or rabbit IgG (not shown) and resolved by 10% (reducing) or 7.5% (nonreducing) SDS-PAGE in the presence (+) or absence (−) of β-ME followed by immunoblot analysis with anti-MT6-MMP mAb1142. Asterisk in E shows a nonspecific band. F, human PMN lysate was mixed with Laemmli SDS-sample buffer with (+) or without (−) β-ME and resolved by 7.5% SDS-PAGE followed by immunoblot analysis with mAb1142. Arrowhead and arrow in A–F indicate the ∼120- and 57-kDa species of MT6-MMP, respectively.