Expression of VNUT in mouse neutrophils.
A, neutrophils from the bone marrow of WT and VNUT−/− (KO) mice were stained with a Giemsa stain. Segmented nuclei were stained purple. Bar, 10 μm. B, RT-PCR analysis was performed using total RNA isolated from bone marrow-derived neutrophils of WT and KO mice (523 bp) after RT reaction (+RT) and without RT reaction (−RT). The PCR product from VNUT cDNA was shown as a positive control. The expression of mouse glyceraldehyde-3-phosphate dehydrogenase (mG3PDH) was also shown for internal RNA quality control (150 bp). C, Western blot of bone marrow-derived neutrophil membrane vesicles (100 μg) prepared from wildtype and VNUT−/− mice and probed using anti-VNUT antibody. The position of VNUT (70 kDa) is marked with an arrowhead. The expression of V-ATPase subunit A, FPRL1, and VAMP2 is also shown. D, indirect immunofluorescence microscopy revealed that VNUT is expressed in wildtype mouse bone marrow-derived neutrophils. No VNUT immunoreactivity was observed in neutrophils from VNUT−/− mice. Inset, background signal with preabsorbed anti-mouse VNUT antibody. Bars, 10 μm.