Fig. 9

Fig. 9 HKi2 treatment increases Akt phosphorylation at S⁴⁷³ that is dependent on PI3K activity. (A-E) hCMEC/D3 cells treated with HKi2 (50 μM) or vehicle control. Cells lysates were analyzed by western blot for Akt and p-Atk S⁴⁷³ protein levels. (A) HKi2 treatment for 1 h activated Akt signaling. (B) HKi2 treatment for 4 h activated Akt signaling and an inactive compound (50 μM), 2-hydroxy-1-naphthoic acid, did not (compound 9). (C) HKi2 treatment for 24 h activated Akt signaling, but the total levels of total Akt were going down. (D,E) Blocking the kinase activity of (E) Akt with MK-2206 (20 μM) and (F) PI3K with LY294002 (10 μM) blunted the activation of Akt by HKi2. (A-H) Bar graphs represent average ± SEM (n = 4), with (A,C) t test, and (B,D,E) one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.