Publication protocol
The cell line samples were homogenized with ice-cold RIPA lysis buffer that was added with protease inhibitor (Complete EDTA-free, #10634200, Roche) and phosphatase inhibitor (PhosSTOP, #04906837001, Roche) and centrifuged at 20,000 x g for 30 minutes at 4°C. Supernatants were collected and kept in -80°C. Bradford method was used to quantify the protein concentration required for western blot sample loading (Pierce BCA Protein Assay Kit, #23225). All samples were dissolved in 1X LDS sample buffer and 1X reducing agent (Life Technologies) and heated for 5 minutes at 95°C. An equal concentration of protein was electrophoresed and separated in NuPage 4-12% Bis-Tris gel (Invitrogen, #NP0322) and transferred to a 0.2 um nitrocellulose membrane (#IB301032) using iBlot (Invitrogen). Blocking was carried out in 5% bovine serum albumin, 0.1% Tween 20 in PBS for non-specific binding for an hour at room temperature. The membranes were incubated overnight at 4°C with mouse monoclonal primary antibodies: OPN, LFMb-14 (1:200, sc-73631, Santa Cruz biotechnology); anti-flag (1:2000, F1804, Sigma), MMP9 2c3, (1:500, sc-21733, Santa Cruz Biotechnology), Anti-HS100A8 (1:500, MAB4570, R&D Systems,) and normalized with actin, C4 (1:5000, sc-47778, Santa Cruz Biotechnology). They were washed and incubated for 60 minutes at room temperature with secondary antibody, goat anti-mouse (1:5000, HRP, sc-2055, Santa Cruz Biotechnology). The membrane was visualized by chemiluminescent HRP substrate reagent (1:1) (Immobilon Western, WBKLS0500, Millipore) using ChemiDoc MP Imaging System (Bio Rad).
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