Fig 2: AKT1 is weakly expressed in microglia but increases following brain injury. (A) Upper and lower panels: Representative images of AKT1 (red) costaining with the microglial marker IBA1 (blue) and astrocytic marker GFAP (green) in area CA1 at high magnification. Profile views of the Z-stacks through IBA1+ cells show low levels of AKT1 in microglia cell bodies but not astrocytes. (B) High magnification image of AKT2 (red) costaining with IBA1 (blue) and ALDH1L1 (green) in area CA1 shows AKT2 colocalization with the astrocytic but not microglial marker. Profile views of the Z-stacks through an IBA1+ cell confirm absence of AKT2 in the microglial cell body. (C) High magnification image of AKT3 (red) costaining with IBA1 (blue) and ALDH1L1 (green) in area CA1 shows absence of AKT3 from both microglia and astrocytes. Profile views of the Z-stacks through an IBA1+ cell confirms that AKT3 is not detected in the microglial cell. (D) IBA1 (green) staining in hippocampal area CA1 following brain injury shows activation of microglia near the injury site, indicated by their ameboid morphology; arrowheads denote the wound track. Costaining with AKT1 (red) and counterstaining with NeuN (blue) show increased AKT1 signal in microglia compared with uninjured brain tissues (A). (E) Higher magnification of the white square in (D) with profile views of the Z-stack images through an IBA1+ cell (arrow) confirms colocalization with AKT1 immunostaining.

Fig 3: AKT isoform expression in human brain tissue. Immunofluorescent staining using isoform-specific AKT antibodies and cell-type-specific markers in human brain tissue isolated from the temporal cortex shows an expression pattern like our mouse brain staining. AKT1 (red, left column) shows colocalization with the neuronal marker NeuN (green), AKT2 (red, middle column) shows colocalization with the astrocytic marker GFAP (white), and AKT3 (red, right column) shows diffuse staining in neuronal cell bodies and neuropil throughout the tissue. Bottom row: Human cortical tissue costained for NeuN (green) and GFAP (white) with AKT isoform-specific primary antibodies omitted but the secondary antibody included (red) to demonstrate the specificity of AKT isoform staining. Hoechst (blue), nuclear stain. Specimens were obtained from four subjects (N = 2/sex) considered as controls by the brain bank with a mean age of 58.75 years (Table 1), and images are representative of AKT isoform staining patterns for each subject.

Fig 4: Insulin induces changes to the electropherograms of Akt2 and Akt3. Electropherograms of cIEF immunoassays of (A) Akt1, (B) Akt2, (C) Akt3, (D) p-Akt (Thr308), (E) p-Akt (Thr450), and (F) p-Akt (Ser473) following treatment with insulin for 30 min. Arrows point to new peaks that appeared after treatment with insulin. The dashed line highlights the distribution of p-Akt (Thr450) as a function of pI values. (G) Capillary Western (CW) immunoassays of p-Akt (Thr308), p-Akt (Thr450), and p-Akt (Ser473) before and after treatment with insulin. Pan-Akt and ß-actin served as the loading controls. (H) Relative expression level of p-Akt (Thr308), p-Akt (Thr450), and p-Akt (Ser473) before (solid black) and after (textured) treatment with insulin. Error bars indicate standard deviations across six repeated measurements using CW immunoassays per experimental condition. Asterisks indicate a statistical significance of p = 0.01 versus untreated control.

Fig 5: Cell-type expression of AKT isoforms in the amygdala mirrors the expression patterns in hippocampus and cortex. (A, B) Representative images of the amygdala costained for AKT1 (red) with the interneuronal markers (A) PV (green) and (B) GAD67 (green). PV is more prominently expressed in the BLA (left outline) compared with the CeA (right outline), but GAD67 staining shows that interneurons are present in both subregions of the amygdala. AKT1 is also present in both subregions. (C) Higher magnification images of AKT1 staining in the LA corresponding to the white square in (A) shows that PV is detected in cells positive for AKT1 (white arrows, left column), whereas the astrocytic marker ALDH1L1 is not detected (white arrows, right column). (D) Higher magnification images of the amygdala corresponding to the white square in (B) shows AKT1+ cells (white arrows) with perisomatic GAD67 staining in the CeA. (E) Representative image of AKT2 staining in the BLA. (F) Higher magnification of the LA corresponding to the white square in (E) shows that AKT2 colocalizes with ALDH1L1 staining (white arrows). (G) Representative image of AKT3 staining in the BLA. (H) Higher magnification of the LA corresponding to the white square in (G) shows that PV+ interneurons (white arrows, left column) do not express AKT3. Costaining with ALDH1L1 shows that ALDH1L1 also is not detected in cells positive for AKT3 (white arrows, right column) in theLA.