Following transfer of the separated proteins to a membrane, the latter was blocked using phosphate-buffered saline-1% casein and incubated with the intact mAbs 9E8 or 3G4 followed by the donkey anti-mouse horseradish peroxidase-conjugated immunoglobulin G (Jackson ImmunoResearch, West Grove, PA, USA) and a TMB/M substrate (SurModics, Eden Prairie, MN, USA). Protease activity assay The cleavage assays were performed in triplicate in wells of a 96-well plate using the purified wild-type catalytic domain name of MT1CMMP (10?nM) and the fluorescent peptide MCA-PLGL-Dpa-AR-NH2 substrate in 0.2?ml 50?mM HEPES, pH 6.8, containing 1?mM CaCl2, 0.5?mM MgCl2 and 10?M ZnCl2. 31 The MT-loop MT1CMMP mutants The MT-loop mutants were obtained by PCR using the E240A mutant sequence as a template. The mutant constructs were then re-cloned into the pET101 vector (Invitrogen), expressed in em Escherichia coli /em , purified from inclusion body using metal-chelating chromatography and refolded to restore the native conformation. A typical yield of the purified MT1CMMP constructs was 1?mg from 10?mg inclusion bodies. The ability of the mutants to interact with the antibodies was tested using Western blotting. Following the transfer of the separated proteins to a membrane, the latter was blocked using phosphate-buffered saline-1% casein and incubated with the intact mAbs 9E8 or 3G4 followed by the donkey anti-mouse horseradish peroxidase-conjugated immunoglobulin G (Jackson ImmunoResearch, West Grove, PA, USA) and a TMB/M substrate (SurModics, Eden Prairie, MN, USA). Protease activity assay The cleavage assays were performed RMC-4550 in triplicate in wells of a 96-well plate using the purified wild-type catalytic domain name of MT1CMMP (10?nM) and the fluorescent peptide MCA-PLGL-Dpa-AR-NH2 substrate in 0.2?ml 50?mM HEPES, pH 6.8, containing 1?mM CaCl2, 0.5?mM MgCl2 and 10?M ZnCl2. Where indicated, before the cleavage reactions the indicated concentrations of TIMP-2 alone or jointly with mAbs 9E8 or 3G4 were coincubated for 30?min at 20?C with MT1CMMP samples. Initial reaction velocities were monitored constantly at em /em ex RMC-4550 lover=320?nm and em /em em=400?nm on a fluorescence spectrophotometer. Antibody-peptide binding assay Using the MT1CMMPTIMP-2 complex structure (PDB accession 1BQQ) as a guide, we synthesized the 31 10-residue long peptides overlapping by 5 residues (YAIQGLKWQH, LKWQHNEITF, NEITFCIQNY, CIQNYTPKVG, TPKVGEYATY, EYATYEAIRK, EAIRKAFRVW, AFRVWESATP, ESATPLRFRE, LRFREVPYAY, VPYAYIREGH, IREGHEKQAD, EKQADIMIFF, IMIFFAEGFH, AEGFHGDSTP, GDSTPFDGEG, FDGEGGFLAH, GFLAHAYFPG, AYFPGPNIGG, PNIGGDTHFD, SAEPWTVRNE, TVRNEDLNGN, LEHSSDPSAI, DPSAIMAPFY, MAPFYQWMDT, QWMDTENFVL, ENFVLPDDDR, PDDDRRGIQQ, RGIQQLYGGE, LYGGESGFPT and SGFPTKMPPQ). The peptides, when combined, represented the molecular surface of the catalytic domain name of human MT1CMMP. Peptides (1?g each) were spotted on a nitrocellulose membrane (Bio-Rad). The membrane was blocked using phosphate-buffered saline-1% casein (30?min, 20?C), incubated for 2?h with mAbs 3G4 and 9E8 (0.5?g/ml each), extensively washed in phosphate-buffered saline-0.05% Tween-20 and then incubated for 1?h with the donkey anti-mouse horseradish peroxidase-conjugated immunoglobulin G. The immunoreactive peptide spots were visualized using a TMB/M substrate. As a control, we also synthesized and tested 20 ten-residue peptides overlapping by 5 residues (EGDLKWHHHN, WHHHNITYWI, ITYWIQNYSE, QNYSEDLPRA, VTPLTFTRVY, FTRVYSRDAD, SRDADIVIQF, IVIQFGVAEH, GVAEHGDGYP, GDGYPFDGKD, AFPPGPGIQG, DDELWSLGKG, SLGKGQSYSL, ALGLDHSSVP, HSSVPEALMY, EALMYPMYRF, PMYRFTEGPP, TEGPPLHKDD, LHKDDVNGIR and VNGIRHLYGG), which, when combined, represented a significant portion of the molecular surface of the human MMP-9 catalytic domain name (PDB accession 1GKC).32 To calculate the intensity of the reactive spots, the images were scanned and digitized. MMP-2 activation assay The status of MMP-2 was analyzed using gelatin zymography of the serum-free medium aliquots (15?l). Where indicated, fibrosarcoma HT1080 cells (1 105 seeded in wells of a 48-well plate) were stimulated for 24?h with phorbol 12-myristate 13-acetate (50?ng/ml) alone and also in the presence of GM6001 (10?M), TIMP-1 (100?nM), TIMP-2 (5C100?nM) and mAbs 3G4 or 9E8 (5C70?nM each). Cell invasion assay Assays were performed in wells of a 24-well, 8?m pore size Transwell plate (Corning, Corning, NY, USA). A 6.5?mm place membrane was coated with 0.1?ml rat tail type I collagen (0. 1?mg/ml; BD Biosciences, Franklin Lakes, NJ, USA) and then air dried for 16?h. The collagen covering was rehydrated for 1?h in 0.1?ml Dulbecco’s Modified Eagle medium. The inner chamber contained 0.6?ml Dulbecco’s Modified Eagle mediumC10% fetal bovine serum as a chemoattractant. Where indicated, mAb 9E8 (100?nM) was added to both inner and outer chambers. Cells (1 105 in 0.1?ml serum-free Dulbecco’s Modified Eagle medium) were allowed to migrate for 3.5?h at 37?C in a CO2 incubator. The cells remaining on the top surface of the membrane were removed with a cotton swab. The cells on the bottom surface of the membrane were fixed and stained for 10?min using 0.5?ml 0.2% crystal violet Rabbit Polyclonal to ARF6 in 20% methanol. The incorporated dye was extracted using 0. 3?ml 1% SDS and the A570 was measured using a plate reader. Data are RMC-4550 means s.e. from three individual experiments performed in RMC-4550 triplicate. Cell invasion levels were calculated relative to the untreated wild-type HT1080 cells RMC-4550 (=100%). Molecular modeling In our modeling studies, we used the structures of the anti-Ras antibody (PDB accession 2UZI)33 and the catalytic domain name of MT1CMMP complexed with TIMP-2 (PDB accession 1BQQ).34 To estimate the space occupied by the antibody, the putative complex of the MT1CMMP catalytic domain with mAb 9E8 was modeled using ZDOCK,35 and 1BQQ and 2UZI as templates. Because anti-MT1CMMP mAbs 9E8 and DX240014, 15 bind the comparable regions of MT1CMMP, we also replaced em in silico /em , using Modeller,36 the residue positions in the complementarity determining regions in the light and heavy chains.