Figure 1.

Loss of Rictor significantly suppresses Akt and mammalian target of rapamycin complex 2 (mTORC2) signaling in peritoneal macrophages, reduces p-Akt S⁴⁷³ in monocytes and neutrophils in vivo, and decreases blood leukocyte numbers. (A–C) Peritoneal macrophages were isolated from Rictor^fl/fl and MRictor^−/− mice (n = 3/group), incubated overnight in serum-free media then untreated or treated with insulin (100 nM) for 10 and 15 min. Proteins were extracted, resolved (60 μg/well) and analyzed by western blot using the antibodies as indicated. Note the reduction of p-Akt S⁴⁷³, p-Akt³⁰⁸ and mTORC2 downstream signaling, p-NDRG1 and p-PKCα in MRictor^−/− macrophages compared to control Rictor^fl/fl cells. Graphs represent data (mean ± SEM; *p < 0.05 compared to control untreated Rictor^fl/fl cells). (D–G) Representative plots of gaiting strategy to analyze Akt S⁴⁷³ phosphorylation levels in bone marrow cells of Rictor^fl/fl and MRictor^−/− mice in vivo (D,E). Note MRictor^−/− bone marrow neutrophils (F) and monocytes (G) expressed significantly less p-Akt S⁴⁷³ (red) than control Rictor^fl/fl cells (green) but were higher than the isotype control antibodies (gray). (H,I) Multicolor flow cytometry analysis of bone marrow (H) and blood cells (I) isolated from Rictor^fl/fl (green) and MRictor^−/− (red) mice. Note the increase of T-cells and neutrophils in bone marrow and decrease of white blood cells, B- and T-cells and monocytes but not granulocytes in blood (mean ± SEM; *p < 0.05 compared to control sample). These experiments were repeated three times.