Figure 4.

Blockade of RANKL-triggered integrin αvβ3 induction (a) and c-Src-Pyk2-c-Cbl signaling (b) by phloretin, and inhibition of Pyk2 induction in ovariectomized (OVX) mouse femoral bone tissues (c). Raw 264.7 macrophages were cultured in α-MEM and exposed to 50 ng/mL RANKL for 5 days in the absence and presence of 1–20 μM phloretin. OVX mice were orally treated with 10 mg/kg/day phloretin daily for 8 weeks. Cell lysates were subject to SDS-PAGE and Western blot analysis with a primary antibody against integrin αv, integrin β3, c-Src, phospho-Pyk2, and c-Cbl. Representative blot data were obtained from three independent experiments, and β-actin protein was used as an internal control. The bar graphs (mean ± SEM) in the bottom panels represent quantitative results of blots obtained from a densitometer. Respective values not sharing a common letter are different at P < 0.05. Immunohistochemical staining was conducted by using a primary antibody of mouse Pyk2 (brown spot staining) and by counterstaining with methyl green. Representative images were visualized under light microscopy (3 separate experiments). Magnification: 40-fold.