Figure 1

Phenotyping of SKIP ^−/− mice.

(A) SKIP^−/− mice do not express SKIP. Extracts from HeLa cells and peritoneal macrophages (PM) prepared from C57BL/6 or SKIP^−/− mice were examined for the presence of SKIP by Western blotting. Actin expression was used as a loading control. (B,C) SCVs accumulate kinesin-1 in the absence of SifA or SKIP. Mouse embryonic fibroblasts prepared from C57BL/6 or SKIP^−/− mice were infected with GFP-expressing wild type (WT) or ∆sifA strains of Salmonella. Cells were fixed 16 h post-infection and immunostained. (B) Cells were imaged by confocal microscopy for bacteria (green), kinesin-1 (red) and nuclei (light blue). Magnified insets showing single grayscale images for kinesin-1 are presented. (C) Percentages of kinesin-1-positive SCVs. Results are the means ± SD of three independent experiments. (D–F) SKIP is not essential for LAMP1 recruitment to the SCV. BMMs were prepared from C57BL/6 or SKIP^−/− mice and infected with wild type or ∆sifA strains of Salmonella expressing GFP from a plasmid and SseJ-2HA from the chromosome. Cells were fixed 16 h post-infection and immunostained. (D) BMMs were imaged by confocal microscopy for bacteria (white), SseJ (green), LAMP1 (red) and nuclei (light blue). Magnified insets showing single grayscale images for bacteria, SseJ and LAMP1 are presented. (E & F) Percentages of SseJ-positive (E) or LAMP1-positive (F) SCVs. Results are the means ± SD of three independent experiments. (B & D) Bar, 20 μm or 10 μm for the magnified insets. (C,E & D) Unpaired t-tests were used to compare two values. P values: ns, not significant; **P < 0.01; ***P < 0.001.