Figure 7: AKT-1 phosphorylates OSM-9 in vitro.
From: Promotion of behavior and neuronal function by reactive oxygen species in C. elegans
(a) In vitro production of GST fusion proteins of OSM-9. The N-terminal fragment of wild-type and point mutant (T10A) of OSM-9 was fused to GST, expressed and purified from bacterial strain BL21. Shown is a SDS-PAGE gel stained by Coomassie blue. The pan-kinase substrate MBP and GST alone were also shown. (b) AKT-1 phosphorylates OSM-9 in an in vitro kinase assay. FLAG-tagged AKT-1 was transfected in HEK293T cells. After pull-down by anti-FLAG affinity beads, AKT-1 was tested for its ability to phosphorylate purified MBP, GST, and GST fusion proteins using (γ-32P)ATP as the substrate. Top panel: autoradiograph. Bottom panel: Western blot showing the amount of AKT-1 used in each experiment. (c) A schematic model illustrating the signaling pathway that mediates H2O2-induced potentiation of neuronal activity. Question marks indicate that additional substrates may exist.
