Mouse anti flag ab

Recombinant murine M-CSF was obtained from R&D Systems. Glutathione-S-transferase (GST)-RANKL was expressed in our laboratory. The GST-RANKL vector was constructed by PCR cloning a SalI/NotI fragment corresponding to amino acids 158–316 of murine RANKL from osteoblast cDNA (forward primer, 5′-ATGTGGCCCGTCGACGCAAGCCTGAGGCCCAGCC-3′; reverse primer, 5′-AAACATCTAGGGCGGCCGCGCTAATG-3′) and cloning into pGEX-4T-1 (GE Healthcare). Both strands were sequenced. High-expressing BL21 (Stratagene) cells were lysed under nondenaturing conditions and GST-RANKL was purified over a glutathione-Sepharose column followed by ion exchange chromatography (Lam et al., 2000 (link)). The sources of antibodies were as follows: rabbit anti-integrin β3 (Cell Signaling Technology); mouse antiphosphotyrosine (4G10), rabbit anti-FcRγ from Millipore; mouse anti-NFATc1 and rabbit anti-Syk (N19; Santa Cruz Biotechnology, Inc.); mouse anti-Flag Ab, rabbit anti-Flag Ab, mouse anti-Talin Ab, and mouse anti-actin Ab (Sigma-Aldrich); mouse anti-HA Ab (Covance); goat anti-OSCAR Ab (R&D Systems); rabbit anti-β1 Ab (Abcam). All other chemicals were obtained from Sigma-Aldrich.