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Fig. 3. Nod2 association with actin cytoskeleton. (A) Distribution of the Nod2 protein between Triton-X-100-soluble and -insoluble fractions. HEK293 cells were transfected with 500 ng pcDNA3 and 50 ng of plasmid expressing the HA-tagged Nod2 protein. Twenty-four hours post transfection, cells were treated or not with CytD 50 µM for 1 hour before being harvested for the preparation of Triton-X-100-soluble (S) or -insoluble (I) fractions. Each extract (S, I) was taken from equal number of cells and was separated by SDS-PAGE (10%) followed by western blotting with a rat monoclonal anti-HA or a mouse monoclonal anti-
-actin antibody. (B) Colocalization of ectopically expressed Nod2 with some specific structures of actin cytoskeleton. HEK293, Vero, Mf4/4 cells were transfected with Nod2-expressing plasmid (1 µg). Twenty-four hours post transfection, cells were stained for Nod2 with the rabbit serum (A,D,G) and for F-actin with TRITC-phalloidin (B,E,H). Images were obtained by confocal microscopy. (C,F,I) Merged F-actin and Nod2 images from panels A and B, D and E, G and H, respectively; areas of colocalization are shown in yellow. (C) Colocalization of endogenous Nod2 with some specific structures of the actin cytoskeleton. HT-29 cells were stained for Nod2 with a mixture of both rat anti-Nod2 monoclonal antibodies 7E11 (1:50) and 6F6 (1:50) and for F-actin with TRITC-phalloidin. A negative control (-) without primary antibodies was performed to test for secondary antibody cross-reaction. DIC, differential inference contrast. Diagrams depict the intensity of the fluorescence for each staining along the lines drawn on the overlay images. Arrows indicate areas of colocalization.
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