Figure 6. A-loop Phosphorylation and PMK-1 Are Essential for Induction of Immune Effector mRNAs by DKF-2.

A shows survival curves for the indicated mutant and transgenic animals upon feeding with PA14. B, amounts of 17 mRNAs were quantified (qRT-PCR) in animals expressing dkf-2::DKF-2-GFP in WT or pmk-1(km25) null backgrounds. Fold-induction is: mRNA concentration in a specified transgenic animal ÷ mRNA concentration in WT C. elegans. C shows effects of DKF-2(A⁹²⁵A⁹²⁹)-GFP and DKF-2(E⁹²⁵E⁹²⁹)-GFP expression on levels of 5 mRNAs in a dkf-2(pr3) background. qRT-PCR data are expressed as fold-induction. D, 30 μg total protein from the indicated WT, null and transgenic strains of C. elegans were assayed by Western immunoblot analysis. The blot was probed with anti-DKF-2 IgGs; DKF-2 polypeptides were detected via chemiluminescence. E, A Western blot (prepared as described in D) was probed with IgGs directed against the di-phosphorylated A-loop of PMK-1 (Promega). Phospho-PMK-1 was detected by chemiluminescence. 14-3-3 protein is a loading control. Experiments in Fig. 6 were performed 3 times, yielding similar results.