Immunoblotting of MNs stimulated with rhMIF for various time points. (A) MNs were stimulated with rhMIF (50 nM) for time periods of 1 minute to 45 minutes. rhMIF induced a marked increase in Src, Akt, and NFκB phosphorylation in a time-dependent manner compared with nonstimulated cells. This phosphorylation was inhibited by relatively specific chemical inhibitors (A-E). (B) Western blotting was performed with MNs stimulated with rhMIF (50 nM) for 12 hours to examine the up-regulation of VCAM-1 and ICAM-1. For inhibitor studies, MNs were pretreated with PP2, LY, PD, and PDTC for 1 hour prior to stimulating with rhMIF. All inhibitors were used at 10 μM concentration except PDTC (100 μM). rhMIF induced a marked increase in VCAM-1 and ICAM-1 in MNs at 12 hours. rhMIF-induced MN VCAM-1 and ICAM-1 up-regulations were abrogated by the inhibitors of Src, PI3K, and NFκB, but an inhibitor of Erk1/2 did not affect the expression of the adhesion molecules (F-G). Each blot represents 1 of 3 experiments.