Figure 4.

Lyn is associated with CD74-CD44 by MIF treatment and MIF modulated RANKL-induced AP-1 and calcium signaling via activated Lyn. BMM cells from WT, CD74KO and CD44KO mice were cultured for 3 days with M-CSF alone before treated with RANKL (30 ng/ml) and/or MIF (25 ng/ml). (A)The equal cells of BMM (3×10⁶ cells/60mm dish) were lysed and the equal amount of whole cell protein from BMM cultures was immunoblotted for phospho-Lyn, CD44 and CD74. (B) To determine if CD74 and/or CD44 associates with Lyn for MIF signaling, BMM cells were treated with MIF (25 ng/ml) and immunoprecipitated with CD74 Ab and immunoblotted with phospho-Lyn, Lyn and CD44 Ab. (C) Immunoprecipitated with CD44 Ab and immunoblotted with Lyn Ab and CD74 Ab. (D) Immunoprecipitated with Lyn Ab and immunoblotted with CD44 Ab and CD74 Ab. (E-H) BMM cells were cultured with M-CSF alone for 3 days before treated with RANKL (30 ng/ml) and/or MIF (25 ng/ml) for up to 30 min. The each protein sample immunoblotted using specific antibodies for AP-1 pathway (E, F) and Syk-PLCγ cascade (G). β-actin was used as control.