Figure 3 | Cell Death Discovery

Figure 3

From: Akt1 and Akt3 but not Akt2 through interaction with DNA-PKcs stimulate proliferation and post-irradiation cell survival of K-RAS-mutated cancer cells

Figure 3

Targeting Akt inhibits Akt/DNA-PKcs complex formation. K-RAS-mutated A549 cells were transfected with eGFP-PKcs (a.a. 1-421) and mCherry-Akt1. (a) Forty-eight hours after transfection, the cells were treated with DMSO (D) or 10 μM of MK2206 (MK) for 1 h and then irradiated with 4 Gy. The cells were lysed 10 min post-IR and eGFP-DNA-PKcs-N was precipitated as described. The input and bound fractions were subjected to SDS-PAGE and immunoblot analysis, and eGFP was detected in the inputs and under the IP conditions as the loading control. The experiment was performed in two biological replicates. The results from one experiment are shown. (b) The cells were lysed 10 min past-IR and immunoblot analysis was subjected. The phosphorylation of Akt (Ser-473) and PRAS40 (Thr-246) was analyzed by immunoblotting in the whole-cell lysates using phospho-specific antibodies. The blots were stripped and re-probed with the antibodies against Akt1 and PRAS40. Data indicates mean P-Akt (Ser-473)±S.E.M. from three independent experiments. The asterisks indicate a significant inhibition of Akt phosphorylation following pretreatment with MK2206 (5 μM) for 2 h followed by irradiation with 4 Gy (*P<0.05, ***P<0.001, Student's t-test).

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