Figure 4. Localization of p110δ on Golgi... | Rockefeller University Press

Figure 4.

$Figure 4. Localization of p110δ on Golgi membranes. (A) Confocal imaging reveals the diffuse cytoplasmic and Golgi immunostaining of p110δ (red) colocalized with GM130 (green; magnified) in RAW264.7 macrophages activated by LPS for 2 h. p110γ (red) gave mostly cytoplasmic immunostaining. (B) Stacked Golgi membranes prepared from activated RAW264.7 macrophages were incubated in vitro with cytosol and GTP-γS to generate budded carriers. The remnant Golgi membranes, budded carriers, and cytosol were collected by ultracentrifugations. Samples of these fractions were run on gels (25% of cytosol supernatants; whole membrane pellets) and immunoblotted for p110δ and γ-adaptin as a peripheral protein marker of TGN-derived budded carriers and TNF as cargo. (C) Coverslip-adherent RAW264.7 macrophages were stimulated with LPS over an acute time course. Immunostaining for p110δ (red) and GM130 (green) was performed at times 0, 30 min, and 2 h on ripped-open cells that exposed intact Golgi membranes. At least 20 cells were analyzed per three independent experiments. Arrows highlight colocalization of p110δ with GM130. DAPI (blue) labeling depicts nuclei. Bars, 10 µm.$

Localization of p110δ on Golgi membranes. (A) Confocal imaging reveals the diffuse cytoplasmic and Golgi immunostaining of p110δ (red) colocalized with GM130 (green; magnified) in RAW264.7 macrophages activated by LPS for 2 h. p110γ (red) gave mostly cytoplasmic immunostaining. (B) Stacked Golgi membranes prepared from activated RAW264.7 macrophages were incubated in vitro with cytosol and GTP-γS to generate budded carriers. The remnant Golgi membranes, budded carriers, and cytosol were collected by ultracentrifugations. Samples of these fractions were run on gels (25% of cytosol supernatants; whole membrane pellets) and immunoblotted for p110δ and γ-adaptin as a peripheral protein marker of TGN-derived budded carriers and TNF as cargo. (C) Coverslip-adherent RAW264.7 macrophages were stimulated with LPS over an acute time course. Immunostaining for p110δ (red) and GM130 (green) was performed at times 0, 30 min, and 2 h on ripped-open cells that exposed intact Golgi membranes. At least 20 cells were analyzed per three independent experiments. Arrows highlight colocalization of p110δ with GM130. DAPI (blue) labeling depicts nuclei. Bars, 10 µm.