p190RhoGAP is induced by RANKL and targeted to podosomes.
(A) RhoA (left panel) and Rac (right panel) activities were measured by G-LISA. BMMs were cultured in the absence (−) or in the presence (+) of RANKL for 3 days. Cell lysates were subjected to G-LISA assays according to manufacturer’s instructions. Shown is a representative of three independent experiments. (B) BMMs were cultured for 4 days in the absence (−) or in the presence of RANKL. Protein extracts were prepared at day 0, 2 and 4 of RANKL addition, and immunoblotted for p190RhoGAP, P-COF and actin as a loading control. (C) OCs differentiated onto coverslips were stained for actin (red) and p190RhoGAP (green) (upper panels) or for cofilin (green), p190RhoGAP (red), and actin (blue) (lower panels). Panels on the right are merged images. Scale bars: 5 µm. (D) BMMs and pre-osteoclasts (pOC) were treated (+) or not (−) for 1 h with 100 µM Y27. Protein extracts were prepared and immunoblotted for phospho-cofilin (P-COF). Total cofilin was revealed after stripping of the phospho-cofilin antibody to serve as loading control. The P-COF/COF ratio was determined by densitometry using the ImageJ software, and the results are shown at the bottom.