Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 3. Rate of activation of MMP-2 by MT2-MMP via the TIMP- 2-independent pathway. Gelatin zymography of conditioned medium of transfected Timp2/ cells expressing (A) hMT2-MMP cultured in the absence of TIMP-2, or (B) hMT1-MMP cultured in the presence of 3 nM TIMP-2, or vector control cells cultured in the presence of 3 nM TIMP-2 as indicated. Cells were cultured in serum-free medium con- taining 5 nM recombinant TIMP-2-free pro-MMP-2 with and without ConA for the times indicated in hours. Medium containing pro-MMP-2 incubated without cells (M) is shown, and the positions of pro-, inter- mediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Activation Assay, Zymography, Transfection, Expressing, Cell Culture, Plasmid Preparation, Control, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 4. Effects of TIMP-2 and TIMP-4 on MMP-2 activation by MT2-MMP. Gelatin zymography of conditioned medium of Timp2/ cells transfected with: A, hMT2-MMP; B, hMT1-MMP; or C, vector alone. Cells were cultured for 24 h in serum-free medium containing 5 nM TIMP-2-free pro-MMP-2, and between 0 and 250 nM TIMP-2 or TIMP-4 was added as indicated. Medium containing MMP-2 incubated without cells (M) is shown, and the positions of pro-, intermediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Activation Assay, Zymography, Transfection, Plasmid Preparation, Cell Culture, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 5. Activation of MMP-2 by MT2-MMP is cell-associated. Gelatin zymography of cell extracts and culture supernatants of Timp2/ cells expressing hMT2-MMP or hMT1-MMP or transfected with the vector alone. Cells were cultured for 24 h in serum-free me- dium with and without 5 nM recombinant TIMP-2-free pro-MMP-2 and 3 nM TIMP-2 as indicated. Culture supernatants were removed, and cells were washed extensively in PBS before being lysed in 0.2% Zwit- tergent. Medium containing pro-MMP-2 incubated without cells (M) is shown, and the positions of pro-, intermediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Activation Assay, Zymography, Expressing, Transfection, Plasmid Preparation, Cell Culture, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 6. Recombinant MMP-2 hemopexin C domain inhibits ac- tivation of MMP-2 by MT2-MMP. Gelatin zymography of cell culture supernatants of Timp2/ cells expressing: A, hMT2-MMP; B, hMT1- MMP; or C, transfected with the vector alone. Cells were cultured for 24 h in serum-free medium containing a 0- to 500-fold molar ratio of recombinant MMP-2 hemopexin C domain (CD) to pro-MMP-2. Cells expressing hMT1-MMP or transfected with the vector alone were incu- bated in the presence of 3 nM TIMP-2 (B and C). Medium containing MMP-2 incubated without cells (M) is shown, and the positions of pro-, intermediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Recombinant, Zymography, Cell Culture, Expressing, Transfection, Plasmid Preparation, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 7. MT2-MMP does not activate truncated MMP-2 lacking the hemopexin C domain. Gelatin zymography of culture superna- tants and cell extracts of Timp2/ cells expressing hMT2-MMP or hMT1-MMP or transfected with the vector alone. Cells were cultured for 24 h in serum-free medium containing either recombinant full- length pro-MMP-2 or pro-MMP-2CD as indicated. Cells expressing hMT1-MMP or transfected with the vector alone were incubated in the presence of 3 nM TIMP-2 ( TIMP-2). Clones #4.7 and #42 expressing hMT2-MMP were incubated without TIMP-2. Full-length MMP-2 (lane 1) or MMP-2CD (lane 13) supernatants were treated with 0.5 mM p-aminophenylmercuric acetate as activation standards (Std) and to show that both forms of recombinant MMP-2 could be activated. The positions of MMP-2 and MMP-2CD are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Zymography, Expressing, Transfection, Plasmid Preparation, Cell Culture, Recombinant, Incubation, Clone Assay, Activation Assay