Impaired trafficking and secretion of TNF in macrophages from genetically inactivated p110δ mice. (A and B) Supernatants from LPS-stimulated WT and p110δ^D910A/D910A BMM cultures were collected every 2 h over a 10-h time course for ELISA measurements of secreted TNF (A) and IL-6 (B). Results indicate mean ± SEM from three littermates of each genotype. ND, not detected. (C) Flow cytometric analysis of intracellular and surface TNF staining in WT and p110δ^D910A/D910A BMMs treated with LPS and TAPI for 2 h. Mean fluorescence intensity in each TNF channel expressed as mean ratio ± SEM relative to LPS-stimulated WT from three independent experiments. ***, P < 0.001. Representative overlay histograms are shown for LPS-stimulated WT and p110δ^D910A/D910A BMMs. (D and E) Confocal analysis of TNF staining in activated WT and p110δ^D910A/D910A BMMs in the presence (D) or absence (E) of TAPI for 2 h. (D) Surface (red) and intracellular (green) TNF staining. Magnified images in D show BMMs costained with phalloidin (blue) to depict surface actin. (E) Intracellular TNF (green) in BMMs costained with the Golgi marker GM130 (red). Differential interference contrast images are included to define cells. Bars, 10 µm.