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. 2013 Jun 3;8(6):e66209. doi: 10.1371/journal.pone.0066209

Figure 3. Msp impairs Akt localization and activation upon fMLP stimulation.

Figure 3

Akt is a downstream target of PI3-kinase. Akt localization and activation can be considered indirect measures of PIP3 generation. A) Localization of Akt and actin in neutrophils visualized by indirect immunofluorescence. Alexa 594-phalloidin F-actin label in red and Akt antibody label in green. Asterix indicates leading edge of neutrophil. B) Graph represents the percentage of cells in which Akt and actin fluorescence are localized together or mislocalized (mean ± SEM, Msp + fMLP treatment (white bars), fMLP treatment alone (black bars). C) Phosphorylation of Akt in cell lysates measured by immunoblotting, following Msp pretreatment and fMLP stimulation. D) PTEN inhibition with the inhibitor VO-OHpic increased PIP3 production and downstream signaling (left panel graph). Treatment of neutrophils with VO-OHpic prior to Msp treatment restored PIP3 production and Akt signaling in response to fMLP stimulation (right panel graph). Representative phospho-Akt immunoblots are shown in panels B and C. Graphs represent the mean ± SEM densitometry of 3 independent experiments (* P<0.05, ** P<0.01, *** P<0.001, ns =  not significant, unpaired t test).