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. 2009 Dec 23;103(2):1114–1122. doi: 10.1152/jn.00980.2009

Fig. 1.

Fig. 1.

Phosphoinositide-3-kinase (PI3K) localizes to the olfactory cilia and can be activated by odorants. A: Western blot analysis of the catalytic p110 subunit using a pan-specific PI3K antibody (p110pan) and antibodies against p110β and p110γ in rat spleen extract, deciliated olfactory receptor neuron (ORN) membranes, and olfactory cilia-enriched membranes. Data are representative of ≥3 independent experiments. B: immunostaining of rat olfactory epithelium (OE) for p110β (red, top left) and olfactory marker protein (OMP), which stains mature ORNs (green, middle left). Double labeling (merged, middle right) shows p110β is abundantly present in mature ORNs. Preabsorption of the p110β antibody with the respective blocking peptide eliminated the staining to the background level (top right). p110β is colocalized in olfactory cilia with adenylate cyclase 3 (green, bottom left; merged, bottom right). Scale bars: 50 μm (top and middle), 25 μm (bottom). C: total PI3K activity measured as pmol of 3′-phosphorylated inositol lipids per 50 μg of protein with an enzyme-linked immunosorbant assay (ELISA) assay in membranes isolated from the cells dissociated from OE and incubated with Henkel-100 (H100, 10−3 dilution) for 5 s. The level of PI3K activity in the absence of odorant (left bar) was below the detection threshold. 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002, 10 μM; red bar) significantly reduced the odorant-dependent increase in PI3K activity (t-test: *P < 0.05). D: direct detection of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) using an overlay dot blot. Left: schematic of the actual blot shown on the right. Data shown are representative of 4 independent experiments. Incubating olfactory ciliary membranes with H100 (10−3 dilution) for the indicated period of time transiently elevated levels of PIP3 at 2 and 5 s (lane 1). PIP3 standards (lane 2) were used to estimate the absolute concentration generated. The inability of the probe to detect different from PIP3 phospholipids (lane 3) controlled for the specificity of the method.