RKIP blocks JAK and Raf-mediated STAT3 activation.

<p>(A) RKIP inhibits JAK1, 2-triggered STAT3 activation. 293T cells were transfected with the Luc reporter construct containing 2× STAT3 binding SIE-fragment and co-transfected with either JAK1, 2, 3 or TYK2 in the presence or absence HA-RKIP and ENMD-1198. Cells were placed in serum free medium and cell extracts were subjected to luciferase assay to estimate transcriptional activity. Averages and standard deviations (error bars) of the values of luciferase activities from triplicates of 3 representative experiments are shown. (B) Western blot analysis demonstrating inhibition of STAT3 pY⁷⁰⁵ phosphorylation in HA-RKIP and JAK1 or 2 transiently transfected 293T cells. Raf-mediated STAT3 pY⁷⁰⁵ phosphorylation is inhibited by RKIP: (C) 293T cells were transfected with Raf-BXB, CMV empty vector or HA-RKIP. After 48 h cells were left untreated or exposed to IL-6 for 1 h. Cell lysates were analyzed by Western blot. (D) 293T cells were transfected with the STAT3 luciferase reporter construct and/or with EV, Raf-BXB or HA-RKIP. After 24 h, some cells were treated with PD98059 (PD) and after 48 h from the initial time of transfection, some cells were treated with IL-6 for 6 h. A paired t-test was performed indicating *(p<0.000005) increase in BXB transfected cells when compared to EV; decrease in STAT3 transcriptional activity **(p<0.00006) in PD treated cells transfected with BXB and ***(p<0.00004) in RKIP and BXB transfected cells when compared to the cells transfected with BXB. Luciferase reporter activities for triplicates of 3 representative experiments +/- s.d. are shown. Western blot analysis of cells transfected with the indicated proteins in the presence or absence of PD treatment.