Journal: The Journal of biological chemistry

Article Title: The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity.

doi: 10.1016/j.jbc.2023.103048

Figure Lengend Snippet: Figure 2. Inhibition of ADAMTS1 by TIMPs. A, inhibition of versicanase activity by TIMP family members. TIMP1, TIMP2, TIMP3, and TIMP4 (each at 500 nM) were incubated with ADAMTS1 (100 nM) for 1 h at 37 C before addition of V1-5GAG and digestion for 2 h. Following SDS-PAGE under reducing conditions (5% β-mercaptoethanol) and immunoblotting, FL V1-5GAG and versikine (VSK) were detected by the anti-Vc antibody. A representative immunoblot is shown (n = 2 independent experiments). B, inhibition of peptidolytic activity. TIMPs (each 25 nM) were incubated with a nominal con- centration of 25 nM ADAMTS1 for 1 h at 37 C before addition of the QF peptide substrate fluorescein-5(6)-carbonyl-Ala-Glu-Leu-Asn-Gly-Arg-Pro-Ile-Ser-Ile- Ala-Lys (5(6)-TAMRA) (3.5 μM) and digestion for 2 h. Following subtraction of the background (reactions not containing ADAMTS1), values were converted into percentage of ADAMTS1 activity in the absence of TIMPs and reported as average ± SD (n = 3, each point representing a technical replicate), p < 0.05 by Mann-Whitney test. C, titration of ADAMTS1 with TIMP3. TIMP3 (0–16 nM) was incubated with ADAMTS1 (20 nM nominal concentration) at 37 C for 1 h, and residual activity against the QF peptide FAM-AELNGRPISIAK-Tamra (3.5 μM) was determined. A representative titration curve is shown, each point representing a mean of two technical replicates. Final concentration of ADAMTS1 following titration was 10 nM. FL, full-length; No E, no enzyme; No I, no inhibitor; QF, Quenched-Fluorescent; TIMP, tissue inhibitor of metalloproteinase.

Article Snippet: Semiquantitative proteoglycan cleavage assays Purified V1-5GAG (100 nM) was digested with ADAMTS1, in TNC-B buffer at 37 C for 2 h. Where indicated, 500 μM recombinant human TIMP1, TIMP2, TIMP3, or TIMP4 (R&D Systems, Cat. n.: 970-TM, 971-TM, 973-TM, 974-TSF) were preincubated with 100 nM ADAMTS1 for 1 h at 37 C before digestion.

Techniques: Inhibition, Activity Assay, Incubation, SDS Page, Western Blot, MANN-WHITNEY, Titration, Concentration Assay

Journal: The American Journal of Pathology

Article Title: Matrix Metalloproteinase-1 Produced by Human CXCL12-Stimulated Natural Killer Cells

doi: 10.2353/ajpath.2006.050676

Figure Lengend Snippet: CXCL12 enhances NK-cell invasion and degradation of type I collagen. A: NK cells were assayed for invasive activity in transwell chambers precoated with type I collagen gel (200 μg/ml). CXCL12 (200 ng/ml) was added to the bottom chambers. For inhibiting NK-cell invasion, MMP inhibitors GM6001 (20 μmol/L) or TIMP-2 (200 ng/ml) were added to both the cell suspension and the collagen gel during invasion assays for 20 hours. Results are shown as the mean number of invasive cells in 10 randomly selected fields in a single representative experiment of four performed. **P < 0.001 compared with unstimulated NK cells. B: Glass slides were coated with 25 μg/ml of quenched fluorescent substrate DQ-collagen I. Fresh NK cells were incubated in the presence or absence of 20 ng/ml CXCL12 for 20 hours. Cells were cultured on DQ-collagen I-coated plates for an additional 4 hours. Unstimulated NK cells (a, b, c, and m), CXCL12-stimulated NK cells (d, e, f, and n), and CXCL12-stimulated NK cells with MMP inhibitors GM6001 (20 μmol/L) (g, h, i, and o) or TIMP-2 (200 ng/ml) (j, k, l, and p) are shown. Degradation of type I collagen (green fluorescence) was detected by confocal microscopy (excitation, 488 nm; emission, 530 nm). Pictures were taken at ×40 magnitude (a–l). DIC images are shown. Bar represents 500 μm. The images in the right column (m–p) are shown of the representative degradation of type I collagen by one NK cell taken at ×100 magnitude.

Article Snippet: Recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) was purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Activity Assay, Incubation, Cell Culture, Fluorescence, Confocal Microscopy