Ab92964

BMDMs were cultured on Corning Osteo Assay surface plates (Corning, cat# CLS3987) with osteoclastogenic induction medium (10% FBS, 1% PSN, M-CSF (30 ng/ml), and RANKL (30 ng/ml)). After incubation for 12 days with or without rOPN (0.5 μg/ml, Abcam, cat# ab92964), the cells were removed using 4% NaClO, and then the plate was washed with deionized water^{56 (link)}. Pits were observed as bright spots on the slides under a microscope (ZEISS AxioPlan2, Germany), and the bone erosion area was calculated within ImageJ (version 1.52 v, USA).

Recombinant osteopontin was purchased from R&D Systems (Minneapolis, MN, USA, #1433-OP-050/CF), ACROBiosystems (Newark, DE, #OPN-H5227), and Abcam (Cambridge, UK, #ab92964, #ab281819). Fibronectin, tenascin-C, bone sialoprotein, and vimentin were purchased from R&D Systems (Minneapolis, MN, USA). a-enolase and type II collagen were purchased from Abcam (Cambridge, UK). Fibrinogen was purchased from the Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Antibodies against total focal adhesion kinase (FAK) and phosphorylated FAK were purchased from Cell Signaling Technology (Danvers, MA, USA, #3285 and #8556). Recombinant human tumor necrosis factor (TNF) was purchased from Peprotech (Cranbury, NJ, USA, #300-01A). The secondary antibody used for immunoblot analysis was peroxidase-conjugated anti-rabbit IgG (Agilent, Santa Clara, CA, USA; #P0399).