Monoclonal mouse antibody

All Western blots were run using a Mini-PROTEAN Tetra Cell system (Bio-rad, Hercules, CA). Both 10% and 4–20% gradient polyacrylamide gels were used. For gel electrophoresis, the running buffer was made up of 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS at pH 8.3. Gels were run at constant voltage (200 v) for 30–50 min. For protein transfer to nitrocellulose membranes, transfer buffer was made up of 25 mM Tris, 0.2 M glycine, and 20% methanol at pH 8.5. A constant voltage (90 v) for 60 min was maintained for transfers. The transfer buffer was refrigerated prior to use, and the entire apparatus was kept cold (approx. 4 °C) during protein transfer. Following transfer, blots were incubated based on antibody manufacturer recommendations. For G6PD, GR, and β-actin quantification, blots were incubated for 12 h on a plate shaker at 4 °C with a polyclonal rabbit antibody (1:1000) (Cell Signaling, Danvers, MA) for G6PD, monoclonal mouse antibody (2 µg/mL) (Sigma-Aldrich, St. Louis, MO) for GR, and monoclonal rabbit (1:1000) (Cell Signaling, Danvers, MA) for β-actin. Both anti-rabbit and anti-mouse secondary antibodies (1:1000) were HRP-linked, and an HRP-detection kit followed by photo-development was used (Cell Signaling, Danvers, MA). All G6PD and GR bands were normalized to their corresponding β-actin bands and results are reported as % of β-actin.

After differentiation on glass cover slips, bone marrow-derived osteoclasts (BMOCs) were stained by immunocytochemistry as described previously (Garbe et al., 2012 (link)). For vinculin, talin or paxillin, cells were stained with monoclonal mouse-antibodies (Sigma-Aldrich) and secondary anti-mouse-IgG1-Cy3 (Jackson). A polyclonal rabbit anti-SWAP-70 antibody, produced in our lab and detected using a Cy3-conjugated goat-anti-rabbit antibody (Jackson), was used to assess SWAP-70 expression. Alexa Fluor 488 Phalloidin (Invitrogen) was used to detect F-actin and 4′,6-diamidino-2-phenylindol (DAPI, Roche) for nuclei staining. Samples were mounted with Fluoromount-G (Southern Biotech) and subjected to fluorescent microscopy (Axiophot, Carl Zeiss; Axiovision Software) or confocal microscopy (TCS SP5, Leica).