Site of action for PI3K in TNF secretory pathway. (A–C) LPS-activated RAW264.7 macrophages were incubated with DMSO or 50 µM LY294002 for 2 h, fixed, and immunostained for TNF (red) and, in green, either TfnR (A) or GM130 (B and C). Arrows in A point to multiple TNF-labeled tubules extending from the Golgi complex, and the arrow in B indicates one of these tubules labeled for TNF but not GM130. (C) 3D tomographic reconstructions of confocal z sections (0.48 µm) show multiple discrete TNF-positive tubules (arrows) after treatment with LY294002. DAPI (blue) labeling depicts nuclei. (D) TNF tubules were counted in cells from five fields (40–50 cells/field). Error bars indicate mean ± SEM. **, P = 0.0077. (E) RAW264.7 macrophages were transiently transfected with YFP-p230_GRIP (green) and TNF-mCherry (red) for 2 h and treated with 50 µM LY294002 for 1 h before fixation for confocal microscopy. Arrows indicate two tubules (∼3 and 13 µm in length). (A–C and E) Regions of colocalization or interest are indicated by boxes and are magnified in insets. Bars: (A, B, and E) 20 µm; (C) 10 µm.