FIG 6.

NLRX-1 redistributes in airway epithelial cells following RV infection. (A to F) Polarized 16HBE14o− cells were infected with RV or sham control. After 16 h, the cells were fixed and incubated with a mixture of antibodies to occludin and NLRX-1 (A to D) or a mixture of antibodies to MT-CO1 (mitochondrial protein) and NLRX-1 (E and F). Bound antibodies were detected by secondary antibodies conjugated with either Alexa Fluor 598 (NLRX-1) or Alexa Fluor 488 (occludin [A to D] and MT-CO1 [E and F]). The cells were counterstained with DAPI and subjected to indirect immunofluorescence microscopy. The arrows in panel A represent colocalization (yellow) of NLRX-1 with occludin. Panels C and D are the Z sections of panels A and B, respectively, showing the distribution of NLRX-1 in relation to occludin, and the apical surface of the cultures is marked by the white line. The arrows in panels E and F represent colocalization (yellow) of NLRX-1 with MT-CO1. (G) Cytoplasmic or mitochondrial proteins isolated from sham- or RV-infected cells were subjected to Western blot analysis with antibodies to NLRX-1, MT-CO1, or GAPDH. The absence of a band corresponding to MT-CO1 in the cytoplasmic fraction and the absence of GAPDH in the mitochondrial fraction indicate efficient separation of cytoplasmic proteins from mitochondrial proteins. The images are representative of 3 or 4 experiments.