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. 2015 Jun 22;10(3):1655–1661. doi: 10.3892/ol.2015.3406

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Figure 2. — Rheb phosphorylation induced by Ras overexpression requires PRAK kinase activity. (A) HA-PRAK, HA-PRAK-KM or PRAK-DN were co-transfected with Myc-Rheb and HRas into HEK293 cells. Phosphorylation of Rheb was determined. (B) HA-PRAK, HA-PRAK-KM or HA-PRAK-DN were co-transfected with HRas into HEK293 cells. HA-PRAK was then pulled down and incubated with glutathione S-transferase-Rheb for in vitro kinase assay. Phosphorylation of Rheb was determined. Rheb, Ras homolog enriched in brain; PRAK, p38-regulated/activated protein kinase; KM, kinase-dead mutant HA-PRAK; DN, dominant-negative mutant HA-PRAK; p-, phosphorylated; HA, hemagglutinin; 1/2, 0.1 µg HA-PRAK for DNA transfection; 1/4, 0.05 µg HA-PRAK.