rabbit polyclonal antihuman mmp2  (Cell Signaling Technology Inc)

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Role and Therapeutic Potential for Targeting Fibroblast Growth Factor 10/FGFR1 in Relapsed Rheumatoid Arthritis.

doi: 10.1002/art.42674

Figure Lengend Snippet: Figure 6. Blockade of FGFR1 signaling pathway shows therapeutic effect in RA. (A) Representative pictures to show fibroblast invasion after knocking down FGF10 and FGF7 by siRNA and scramble control (siNC) in pannus FLSs for 40 h in the transwell invasion assay. (B) Scatter plots to quantify inva- sive cells. ns, not significant; ****P < 0.0001. (C) Representative western blots to show MMP1, MMP2, MMP3, and RANKL proteins in pannus FLSs after knocking down FGF10 and FGF7 by siRNA or scramble control (siNC). ACTIN was used as a loading control. (D) WB quantification was performed using the comparative GV method and normalized to the ACTIN expression. ns, not significant; *P < 0.05; **P < 0.01. (E) Representative flow cytometric plots show the frequency of RANKL+ PDPN+ FLSs from synovium (left) and pannus (right) from the same patients with relapse RA. (F) Scatter plots show the accumulated data for RANKL+ PDPN+ FLS of five paired samples from five patients with relapse RA. *P < 0.05. (G) Representative mIHC to show the distribution of TRAP+ CD68+ macrophages in the pannus between patients with relapse and Rem RA. (H) Comparisons of the proportions of TRAP+ CD68+ macrophages in pannus between relapse RA and Rem RA. ****P < 0.0001. (I) Representative photomicrograph of bone slice erosion of pannus cells to show the reduced erosion area by the FGF10 inhibitor PD173074 (50 nM/ml) and the increased erosion area by recombinant human FGF10 (100 ng/ml). (J) Scatter plots to quantify erosion area **P < 0.01; ****P < 0.0001. (K) ELISA assay results show the levels of FGF10 protein in the synovial fluid of patients with relapse RA (n = 9), Rem RA (n = 5), and OA (n = 7); RA in Rem was slightly higher than OA. ns, not significant; ****P < 0.0001. (L and M) Clinical scores (L) and mean paw thickness (M) in the untreated group (NC, n = 8), PD173074 group (n = 8), and MTX group (n = 8). The PD173074 group had significantly reduced clinical scores and joint swelling after 4 weeks of treatment compared with the untreated group; **P < 0.01; ****P < 0.0001. DAPI, 4’,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified eagle medium; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; FGFR, FGF receptor; FLS, fibroblast-like synoviocyte; GV, grayscale value; mIHC, multiplex immunohistochemistry; MMP, matrix metalloproteinase; MTX, methotrexate; OA, osteoarthritis; PDPN, podoplanin; RA, rheumatoid arthritis; RANKL, receptor activator of NF- κB ligand; Rem, remission; siRNA, small interfering RNA; SPSS, stroke-physiological saline solution; TRAP, tartrate-resistant acid phosphatase; WB, western blot. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.42674/abstract.

Article Snippet: Membranes were incubated with antibody dilution–diluted primary antibodies with gentle agitation at 4 C overnight as follows: rabbit antihuman MMP1 antibody (Proteintech; catalog no. 10371-2-AP), rabbit antihuman MMP2 antibody (Proteintech; catalog no. 10373-2-AP), rabbit antihuman MMP3 antibody (Proteintech; catalog no. 66338-1-Ig), and mouse antihuman RANKL antibody (Enzo, catalog no. ALX804-243-C100).

Techniques: Control, Transwell Invasion Assay, Western Blot, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Immunohistochemistry, Small Interfering RNA, Saline