Stimulation of STAT1 serine phosphorylation by RANKL through the p38 MAPK pathway. (A) Time course of STAT1 phosphorylation by RANKL. Osteoclast precursors were serum starved for 12 hours and were stimulated with 100 ng/mL RANKL for the indicated time. Cell lysates were subjected to Western blotting for the phosphorylation of STAT1 at Ser727 and Tyr701 using antiphosphospecific STAT1 antibodies (top and middle rows). The same membrane was stripped and reprobed with anti-STAT1 antibody (bottom row). (B) Serine phosphorylation of STAT1 dependent on RANKL dose. Osteoclast precursors were serum starved for 12 hours and then stimulated with the indicated concentration of RANKL for 10 minutes. Western blot analysis was carried out as described. (C) Effects of p38 inhibitor on RANKL-induced phosphorylation of STAT1. Osteoclast precursors were deprived of serum for 12 hours and were pretreated with 20 μM SB203580, followed by stimulation with 100 ng/mL RANKL for 10 minutes. Cell lysates were prepared and subjected to Western blot analysis, as described.