Multi gauge ver3 x

Real-time qPCR was performed in triplicate for all samples and standards with approximately 25 ng were separated on SDS-polyacrylamide gels (12%) for Western blot analysis using anti-MMP-3, -tissue inhibitor of metalloproteinase (TIMP)-1, -TIMP-2, -TIMP-3, -Wnt5a, -Wnt5b, -Lrp5, -Fzd9, -MMP-1, -MMP-2, -MMP-9, -MMP-13, and -β-tubulin polyclonal antibodies (sc-6839, sc-5538, sc-6835, sc-6836, sc-365370, sc-109464, sc-21390, sc-33509, sc-13595, sc-6840, sc-30073, and sc-9935, respectively; Santa Cruz Biotechnology Inc.), and an anti-MMP-1 antibody (ab118529; Abcam, Cambridge, UK). Visualization and quantification of blotted protein bands were performed using a Multi Gauge-Ver3.X (Fujifilm, Tokyo, Japan).

Cells were cultured for 6 h with or without Poly(P) and then lysed using cell lysis buffer (Cell Signaling Technology Japan, K.K., Tokyo, Japan). Protein lysates were separated on SDS-polyacrylamide gels (12%) in preparation for western blot analysis using anti-ALP, -OC, -OP, -MMP-3, -DMP-1, and -β-tubulin polyclonal antibodies (sc-271431, sc-30044, sc-10593, sc-6839, sc-5538, sc-13595, sc-6840, sc-30073, and sc-9935, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Visualization of blotted protein bands was performed using a Multi Gauge-Ver3.X (Fujifilm, Tokyo, Japan).

Cell proliferation was evaluated using the BrdU-cell proliferation enzyme-linked immunosorbent assay (ELISA; Roche Applied Science, Mannheim, Germany) as described previously (25, 26) . Cells were seeded no significant cross-reactivity with other MMPs (data not shown). Visualization and quantification of blotted protein bands were performed with Multi Gauge-Ver3.X software (Fujifilm, Tokyo, Japan).