Western blot FLS extracts were prepared in RIPA buffer with Complete Protease Inhibitors, denatured in sample buffer and 0. 1 M dithiotreitol, and fractioned on Invitrogen NuPage 4 to 12% precast gels. Following blotting to polyvinylidene despite fluoride membranes and blocking with 5% dry milk, blots were probed with antibodies against phospho or total p38, JNK, Erk, or Akt, as well as with secondary anti rabbit IgG HRP. GAPDH was used as a gel loading control. Membranes were devel oped with Immun Star WesternC ECL substrate and imaged on a VersaDoc imaging system, using QuantityOne software Inhibitors,Modulators,Libraries for image capture and densitometry. Statistical analysis Data are reported as mean and standard error of the mean.

Protein secretion and gene expression data in single time point experiments were analyzed by one way ANOVA followed by Tukey Kramers post hoc test comparing all groups, or by Dunnetts post hoc test com paring control to all others, as appropriate. Time course data were analyzed by two way Inhibitors,Modulators,Libraries ANOVA followed by con trast testing. Students t test was used to examine syner gistic effects of growth factors and cytokines. Real time qPCR data were log transformed prior to analysis. Results Effect of PDGF BB and TGF B on FLS secretion of inflammatory mediators Since PDGF and TGF B are abundant in the rheumatoid synovium, their effect on cytokine induced inflammatory mediator secretion by FLS was examined. TGF B induced only a small amount of IL6, and no effect on IL6 or MMP3 was observed by PDGF BB alone. PDGF and TGF B in combination induced low level secretion of IL6, but not MMPs or chemokines.

The amount of IL6 secreted after 2GF stimulation was comparable Inhibitors,Modulators,Libraries to that observed with TNF as the stimulant. Surprisingly, the two growth factors in combination potently augmented Inhibitors,Modulators,Libraries secretion of IL6 and MMP3 in response to TNF or IL1B. The effect of 2GF was truly synergistic, in that the secretion observed by 2GF and TNF or IL1B in combination was significantly higher than that obtained when adding the values for 2GF alone and cytokine alone. When PDGF BB and TGF B were examined individually, nei ther augmented TNF or IL1B induced MMP3 secretion, and the effect on TNF or IL1B induced IL6 secretion was smaller than that of the growth factor combination. The potentiating effect of 2GF was not simply due to a non specific effect of cell activation, Inhibitors,Modulators,Libraries since the secretion of some but not all mediators was affected.

TNF induced secretion of MMP1 and MCP1 was unal tered by addition of 2GF, and RANTES kinase inhibitor Abiraterone secretion was inhibited, at the same time that IL8 and MIP1 secretion was potentiated along with that of IL6 and MMP3. The effect of 2GF was mediated through activation of growth factor receptors, since the receptor tyrosine kinase inhibitor, imatinib mesylate significantly reversed the potentiating effect of 2GF on TNF induced secre tion of IL6, IL8, MIP1, and MMP3.