QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 4. Both CARDs or LRRs are sufficient to target Nod2 in membrane ruffle-like structures. (A) Colocalization of the wt and mutant Nod2 proteins with F-actin in COS-7 cells. COS-7 cells were transfected with the following amounts of plasmid encoding the HA-tagged or FLAG-tagged wt or mutant Nod2 proteins: pcDNA₃-HA-wt Nod2 and pcDNA₃-HA-CARDs (1 µg each), pcDNA₃-HA-LRRs (4 µg), pcDNA₃-HA-CARDs (0.5 µg), pcDNA3-HA-LRRs (1 µg), pcDNA₃-FLAG-NOD (2 µg). In each case, the total DNA amount was adjusted to 4 µg with pcDNA3. Twenty-four hours post transfection, cells were stained for the wt or mutant Nod2 proteins with anti-HA mAb or anti-FLAG rabbit serum, and for F-actin with TRITC-phalloidin. Images were obtained by confocal microscopy (Leica TCS NT). Arrows indicate areas of colocalization. (B) Distribution of the mutated Nod2 proteins between Triton-X-100-soluble and -insoluble fractions. HEK293 cells were transfected with the following amounts of plasmid encoding the HA-tagged or FLAG-tagged mutant Nod2 proteins: pcDNA₃-HA-CARDs (25 ng), pcDNA₃-HA-CARDs (10 ng), pcDNA3-HA-LRRs (100 ng), pcDNA₃-FLAG-NOD and pcDNA₃-HA-LRRs (2500 ng each). In each case, the DNA total amount was adjusted to at least 2500 ng with pcDNA3. 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 kind of extract (S, I) from equal number of cells was separated by SDS-PAGE (10%) followed by western blotting with anti-HA or anti-FLAG mAbs.

Return to article