MAC1 is necessary for PI3K activation elicited by Aβ. (A) Western blot assays for p110 and PDK levels in membrane fractions of microglia from MAC1+/+ and MAC1-/- mice 10 min after vehicle medium (control group) or Aβ treatment. (B) Enriched microglial cells from MAC1+/+ and MAC1-/- mice were treated with vehicle medium (control group) or Aβ for 10 min, fixed and permeabilized. I and II are MAC1+/+ cultures treated with vehicle or Aβ, respectively. III and IV are MAC1-/- cultures treated with vehicle or Aβ, respectively. (C) Western blot assays for pAKT and AKT levels in microglia from MAC1+/+ and MAC1-/- mice 10 min after vehicle medium (control group) or Aβ treatment. (D) Densitometry was performed with values normalized to respectively loading control and further normalized to control levels. Cells were incubated with a rabbit polyclonal antibody against PIP3 and then with a FITC-conjugated goat anti-rabbit antibody. Shown are representative confocal images. Scale bar: 25 μm. (E) Quantitative analysis of the figures in 6B. Average cellular fluorescence was quantitated from at least 100 separate cellular measurements obtained from each treatment group. Background fluorescence, determined in the absence of fluorescence labeled antibody, was minimal in both cell populations (data not shown). Experiments were performed at least three times. #: p < 0.05 relative to corresponding vehicle-treated control cultures. *: p < 0.05 relative to MAC1+/+ cultures after same treatments.