recombinant human matrix metalloproteinase 2  (Sino Biological)

Journal: Arthritis Research & Therapy

Article Title: Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis

doi: 10.1186/ar2021

Figure Lengend Snippet: Matrix metalloproteinase (MMP)-2 upregulation by macrophage migration inhibitory factor (MIF) is time-dependent. (a) Using gelatin zymography of rheumatoid arthritis (RA) synovial fibroblast culture supernatants, we found MMP-2 upregulation, beginning after 1 hour and increasing continuously over a period of 24 hours. The results represent one of four individual experiments using cells from four donors. (b) Immunofluorescence staining of RA synovial fibroblasts for MMP-2 showed a strong perinuclear and discrete diffuse cytoplasmic expression after 1 hour of stimulation by MIF (50 nM; 400×). Results represent one of four individual experiments using cells from four donors. NS, nonstimulated; pro-MMP, pro-matrix metalloproteinase-2; rhMIF, recombinant human macrophage migration inhibitory factor.

Article Snippet: Molecular-weight marker (Sigmamarker, Sigma) and recombinant human matrix metalloproteinase-2 (rhMMP-2) were used as controls.

Techniques: Migration, Zymography, Immunofluorescence, Staining, Expressing, Recombinant

Journal: Arthritis Research & Therapy

Article Title: Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis

doi: 10.1186/ar2021

Figure Lengend Snippet: Macrophage migration inhibitory factor (MIF)-induced matrix metalloproteinase (MMP)-2 production is PKCδ, JNK, and Src pathway-dependent. Rheumatoid arthritis (RA) synovial fibroblasts were incubated for 6 hours, with or without MIF (50 nM), in the presence or absence of signaling pathway inhibitors: PKC (pan) inhibitor Ro-31-8425 (1 μM), PKCδ isoform-specific inhibitor rottlerin (1 μM), JNK inhibitor JNK II, and Src inhibitor PP2 (10 μM). (a) MMP-2 concentrations in cell culture supernatants were measured by ELISA. Inhibitors to PKCδ, JNK, and Src signaling intermediates inhibited MIF-induced MMP-2 upregulation. (b) Gelatin zymography showed the same effect of these inhibitors on MMP-2 upregulation. Results represent three experiments using RA synovial fibroblasts from six donors. DMSO, dimethyl sulfoxide; JNK, c-jun N-terminal kinase; NS, non-stimulated; pro-MMP, pro-matrix metalloproteinase-2; PKC, protein kinase C; rhMIF, recombinant human macrophage migration inhibitory factor.

Article Snippet: Molecular-weight marker (Sigmamarker, Sigma) and recombinant human matrix metalloproteinase-2 (rhMMP-2) were used as controls.

Techniques: Migration, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Zymography, Recombinant

Journal: Molecular Vision

Article Title: Osmotic induction of cyclooxygenase-2 in RPE cells: Stimulation of inflammasome activation

doi:

Figure Lengend Snippet: Regulation of cyclooxygenases -1 and -2 ( COX1 and COX2 ) gene expression in retinal pigment epithelial (RPE) cells. A : Presence of COX1 and COX2 transcripts in RPE cells. To confirm the correct lengths of the PCR products, agarose gel electrophoresis was performed using products obtained from two RPE cell lines (1, 2) derived from different post-mortem donors. Negative controls (0) were done by adding double-distilled water instead of cDNA as the template. The β-actin ( ACTB ) mRNA level was used to normalize the COX1 and COX2 mRNA levels. B – H : COX1 and COX2 mRNA levels, as determined with real-time reverse transcription (RT)–PCR after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars). The mRNA levels are expressed as folds of unstimulated control. B : Effects of extracellular hyperosmolarity induced by addition of high (+ 100 mM) NaCl on the COX1 and COX2 gene expression levels. C, D : Dose-dependencies of the effects of high extracellular NaCl on the COX1 and COX2 mRNA levels. Ten to 100 mM NaCl were added to the culture medium, as indicated in the bars. The data were obtained after stimulation of the cells for 6 h ( C ) and 24 h ( D ). E : Effect of extracellular hypo-osmolarity (Hypo; 60% osmolarity) on the COX2 gene expression level. F : Effect of the addition of 200 mM sucrose on the COX2 gene expression level. G : Effects of hypoxia induced by cell culture in 1% O 2 , and by addition of 150 µM CoCl 2 , on the COX2 gene expression level. H : Effects of inflammatory and growth factors on the expression of COX2 . The following factors were tested: endothelial growth factor (EGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), placental growth factor-2 (PlGF-2), pigment epithelium-derived factor (PEDF), interleukin-1β (IL-1β), and matrix metalloproteinase-2 (MMP-2). Each factor was applied at 10 ng/ml. Each bar represents data obtained in three to ten independent experiments with cell lines from different donors. Each experiment was performed with cell lines from three to six donors; in total, cell lines from 21 different donors were used for all experiments shown. Statistically significant difference versus unstimulated control: *p<0.05.

Article Snippet: The following compounds were obtained from Calbiochem (Bad Soden, Germany): cyclosporin A, Gö6976, H-89, the hypoxia-inducible transcription factor (HIF)-1 inhibitor, LY294002, human recombinant matrix metalloproteinase-2 (MMP-2), PD98059, PP2, SP600125, SU1498, and U73122.

Techniques: Expressing, Agarose Gel Electrophoresis, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Binding Assay

Journal: Pharmaceutics

Article Title: Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies

doi: 10.3390/pharmaceutics13091390

Figure Lengend Snippet: Example of capillary electrophoresis (CE)-based Pb-Tx cleavage assessment. Electropherograms of CE-based cleavage assessment of the C225-Sub1 Pb-Tx incubated with ( A ) recombinant human membrane-type serine protease 1 (MT-SP1) and ( B ) matrix metalloproteinase-2 (MMP-2) for 4 h. LC, light chain; HC, heavy chain; Pb-Tx, Probody therapeutic.

Article Snippet: Recombinant human membrane-type serine protease 1 (MT-SP1) (3946-SEB), urokinase-type plasminogen activator (uPA) (1310-SE), and matrix metalloproteinase-2 (MMP-2) (902-MP) were from R&D Systems (Minneapolis, MN, USA).

Techniques: Electrophoresis, Incubation, Recombinant, Membrane