Anti mt1 mmp

For RFP-trap assays, MDA-MB-231 cells overexpressing or not MT1-MMPmCh WT or ΔDKV were lysed in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 60 mM beta-glucoside, 10 mM MgCl₂, 1% Triton X-100, and 10% glycerol with anti-protease and anti-phosphatases. RFP-Trap beads (ChromoTek) were added at concentration recommended by the supplier. For in vitro kinase assays, GST-LIMK1 recombinant protein (Abnova) was incubated with precipitated MT1-MMPmCh with RFP-Trap beads (ChromoTek) in stringent conditions (RIPA Buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 10 mM MgCl₂, 1% NP40, 0.5% Na Deoxycholate, 0.1% SDS) as the substrate in the kinase assay buffer containing 50 mM HEPES, 150 mM NaCl, 5 mM MgCl₂, 5 mM MnCl₂, 250 μM ATP for LIMK1. The reaction mix was incubated for 30 min at 30 °C. The reaction was stopped by addition of 2× Laemmli sample buffer. Overexpressed MT1-MMP and phosphorylated bands were respectively determined by SDS-PAGE with an anti-MT1-MMP (Millipore) an anti-phosphotyrosine antibodies (4G10). Immunoprecipitation experiments were replicated at least 3 times except for immunoprecipitation of endogenous MT1-MMP using mouse 2D7 anti-MT1-MMP monoclonal antibody (a kind gift of M.C. Rio and C.L. Tomasetto, IGBMC, Strasbourg, France), which was performed once.