FIG 5.
Pim kinases mediate Jak2-dependent effects on GW182. (A) Western blots examining kinetic changes in expression of GW182, Pim1, or Pim2 in splenic T cells isolated from C57BL/6 mice and activated ex vivo with microbeads coated with anti-CD3 and anti-CD28 antibodies in the presence of 25 U/ml recombinant murine interleukin-2 (rIL-2). GAPDH served as an endogenous control. (B) Western blots for expression of GW182, Pim1, and Pim2 in IL-3-dependent FL5.12 cells expressing either Bcl-xL (xL), myristoylated Akt (mAkt), or the TEL-JAK2 fusion protein (Jak2) following 24 h of culture in the presence or absence of IL-3. Actin served as an endogenous control, and the asterisk denotes a nonspecific band in the Pim1 blot. (C) Effects of chemical inhibitors of the Pim kinases (PIMi, 2.5 μM SGI-1776) or mTOR kinase (0.5 μM Torin1) on expression of GW182 protein and mRNA in FL5.12.Jak2 or FL5.12.mAkt cells. (Top) Representative Western blots for GW182 and Argonaute 1-4 (panAgo) with actin as an endogenous control. (Bottom) Red bars represent mean expression values of the mRNA coding for GW182 relative to the (−)IL-3 condition ± 95% confidence intervals of the means from two independent qRT-PCR experiments performed in quadruplicate. GAPDH was used as the endogenous control. Blue bars represent mean GW182 protein expression values relative to th (−)IL-3 condition ± standard deviations determined by quantification of data from Western blots from three independent experiments. Actin was used to normalize protein loading between lanes. *, P < 0.05.