Figure 5.

Septins localize with lytic granules at the CS. Primary NK cells or their conjugates with 721.221 cells were stained for F-actin (cyan), septin 7, 1, or 2 (red), and perforin (green) to mark lytic granules. Cells were imaged using a confocal microscope in the Airyscan setting. (A) Septins were found closely apposed with perforin-containing lytic granules. Representative z-stack images are shown for septin 7 (top; slice 5 of 11), septin 1 (middle; slice 3 of 12), and septin 2 (slice 6 of 12). Scale bar, 5 µm. Magnified images are ∼1 µm. (B) Accumulation of septin 7 and septin 1 near lytic granules at the CS. Representative z-stack images are shown for septin 7 (top; slice 19 of 39) and septin 1 (bottom; slice 12 of 20). Scale bar, 5 µm; inset scale bar, 2.5 µm. (C) Co-localization of septin 7 with lytic granules at the CS shown in the Airyscan z-stack orthogonal views (slice 14 of 44). Scale bar, 5 µm; inset scale bar, 2.5 µm. (D) Postnuclear (PNL), postmitochondrial (PML), CaCl₂, and CLF were isolated from unstimulated NKL cells as described in the Materials and methods section and immunoblotted as indicated. Immunoblot is representative of three independent experiments.