FIG. 7.

Both catalytic activity and adapter function of PLCγ2 are required for bone resorption in osteoclasts. (A) PLCγ2^−/− BMDM were retrovirally transduced with pMX-PLCγ2, PLCγ2-SH2 (construct carrying the mutations R564K/R672K), or the PLCγ H/F mutant (catalytically inactive construct with mutated H327F/H372F). Cells were grown in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL for 5 to 8 days, fixed, and TRAP stained. TRAP⁺ osteoclasts were observed using a light microscope with a 10× objective. (B) Infected BMDM were lysed and Western blotted (WB) for PLCγ2 expression. Actin expression served as a loading control. (C) Transduced proteins were immunoprecipitated using a FLAG antibody, and production of IP₃ was determined using a radioactive probe. Lipase activity is expressed as percentage of the WT PLCγ2 level, which was given an arbitrary value of 100. (D, E) BMDM infected with pMX-PLCγ2 or coinfected with indicated PLCγ2 mutants plus NFAT2 were plated on dentin and grown in differentiation medium for 10 days. After cells were removed, pits were visualized by staining with peroxidase-conjugated wheat germ agglutinin (E). Area of resorbed bone was determined using Image J software (D). Scale bar, 20 μm. (F) Plcγ2^−/− BMDM were retrovirally infected with Plcγ2 or PLCγ2 mutants SH2 and H/F plus NFAT2 and grown in differentiation medium for 5 to 8 days. Osteoclasts were fixed and immunostained with PLCγ2 antibody.