IL-4 fails to inhibit osteoclastogenesis in RANK-overexpressing cells. (A) Control RAW264.7 cells or RANK-transfected clones 1 and 2 were differentiated to osteoclasts in the presence of rmM-CSF (20 ng/mL) and RANKL (150 ng/mL) ± 10 ng/mL rmIL-4. TRAP-positive multinucleated (more than 3 nuclei) cells were counted. The average number of osteoclasts generated from 3 independent experiments is presented with their respective standard deviation. (B) To ensure that transfected clones were not impaired in IL-4 signaling, growing RAW264.7 (POC) or mature osteoclasts (OC) derived from control, RANK-transfected clone 1 or 2 were stimulated with 0 or 10 ng/mL IL-4 as indicated for 10 minutes, lysed, STAT6 immunoprecipitated, blotted, and probed with an antiphosphotyrosine antibody. To ensure equal protein loading, the blots were stripped and reprobed with anti-STAT6 antibody.