Fig. 2
RIP2 forms a filamentous structure upon activation. a NF-κB promotor activation by domain swap chimeric constructs of RIP2. HEK293T cells were transfected with empty pflag vector, wild-type RIP2, and chimeric RIP2 together with pGL4.32 NF-κB-RE vector and CMV-Renilla vectors. Human NLRC4–CARD (1–100) was used to replace the RIP2–CARD domain (432–540) in human RIP2. Human NLRC4–CARD (E36R) is a monomeric mutant. Error bar represents standard deviation values of three independent repeats. b Tendency of oligomerization of wild-type and chimeric RIP2 proteins. HEK293T cells were transfected with flag-tag wild-type RIP2 or chimeric RIP2 constructs. Total cellular extracts (T) were subjected to 10,000 × g precipitation for 5 min to isolate the supernatant fraction (S) and the pellet fraction (P). A vast majority of RIP2 appeared to be in the pellet fraction. In NLRC4–CARD swapped constructs, a vast majority remained in pellet form. Chimeric constructs with monomeric NLRC4–CARD (E36R) appeared to be mostly soluble. Western blots were representative of three independent experiments. c Negative stain EM images of flag-RIP2, flag-MBP–RIP2, RIP2-kinase, RIP2-kinase-NLRC4–CARD, and RIP2-kinase-NLRC4–CARD (E36R) purified from HEK293T transfected cellular extracts. Flag-RIP2 filaments are about 12-nm thick, similar to RIP2-kinase-NLRC4–CARD. The diameter of flag-MBP–RIP2-fl filaments is wider at 16 nm. No filament was observed for the rest two samples. Scale bar: 100 nm
