Fig. 4

C60 and C77 of Akt are essential for Akt activation in vitro and in vivo. a Representative immunoblots of Akt activation and signalling. 3T3-L1 fibroblasts overexpressing Akt2 -WT, -W80A or -W80A-C60/77S were serum starved and then treated with MK-2206 (10 μM, 30 min) prior to stimulation with insulin. b Quantification of Akt activation and signalling blots. Data are mean ± SEM from n = 3, (***p < 0.001, ****p < 0.0001) by two-way ANOVA. c Expression level of Akt mutants in HeLa cells transfected with indicated vectors for 24 h. d Proliferation of HeLa cell overexpressing Akt2-WT or indicated mutants measured by Hoechst. Data are mean ± SEM from n = 4, **p < 0.01 by one-way ANOVA. e Colony formation assays were performed using HeLa cells overexpressing corresponding Akt2-WT or indicated mutants. Colony numbers were counted and shown as mean ± SEM from n = 8, *p < 0.05 by Brown-Forsythe and Welch ANOVA tests. f Representative images of soft HeLa colonies. g Anchorage-independent cell growth was assessed by soft agar assays using HEK cells overexpressing Akt2-WT or indicated mutants. Colony numbers were counted and shown as mean ± SEM from n = 6, *p < 0.05 by one-way ANOVA. h Representative images of HEK colonies. i Flies overexpressing hAkt2-WT or hAkt2-C60/77S were starved overnight and re-fed for 30 or 60 min. Flies were sacrificed and the samples were immunoblotted using indicated antibodies. j Representative images of flies with depletion of dAkt1 and simultaneous overexpression of human hAkt2-WT or hAkt2-C60/77S. k–m Fly growth were determined by the measurement of body weight (k), body length (l) and wing size (m); ***p < 0.001 by Welch’s t-test. n Flies with depletion of dAkt1 and overexpression of hAkt2-WT or hAkt2-C60/77S were starved overnight and re-fed for 30 or 60 min. Flies were sacrificed and the samples were immunoblotted using indicated antibodies.