Human NK cells contain intracellular stores of AICL associated with the Golgi complex. (A) Representative flow cytometric analyses of intact or permeabilized resting human NK cells (CD3⁻CD56⁺) for cell surface and total AICL expression, respectively, using mAb 7F12 (open histograms). Isotype control stainings are shown in gray. (B) Total AICL protein in resting human NK cells as determined by immunoblotting with mAb 7G4. Cell lysates were treated with either endoglycosidase H or PNGase F before SDS-PAGE, or left untreated (ø). Actin served as loading control. One representative experiment out of 3 is shown. (C) Confocal microscopy revealed predominant colocalization of AICL with the Golgi complex–resident protein Giantin in human resting NK cells. Freshly isolated NK cells were permeabilized and stained with anti-AICL mAb 7F12 (red) in combination with anti-Giantin, anti-LAMP1, or anti-Calreticulin (all green). Nuclei were counterstained with 4,6 diamidino-2-phenylindole (DAPI) (blue). Scale bars, 5 µm. Experiments with NK cells from 3 donors showed similar results. (D-E) Golgi complex–associated localization of AICL in human cell lines. (D) 293 cells stably ectopically expressing an AICL-eGFP fusion protein (green) were stained for Giantin (red) and analyzed by confocal microscopy. (E) U937 cells were costained with anti-AICL mAb 7F12 (green) and anti-Giantin (red). (D-E) Scale bars represent 10 µm.