Fig. 3
Preparation of recombinant RIP2–CARD filament in its signaling active form. a Cartoon illustrations showing how recombinant RIP2–CARD filaments were prepared. The oligomeric seed fraction of SNAP–RIP2–CARD was purified and refolded into functional monomers at resting state. Extended filaments were prepared by coincubating monomers and seeds together. Once extended filaments formed, 3C proteases were used to remove the peripheral SNAP fusion tags. The ordered core RIP2–CARD filaments were revealed afterward. b Negative stain EM images of SNAP–RIP2–CARD seeds (left panel), extended SNAP–RIP2–CARD filaments (middle panel), and the core RIP2–CARD filaments (right panel) at 49,000× magnification. The core RIP2–CARD filaments were much thinner (~7 nm) compared to SNAP–RIP2–CARD filaments (~11 nm). c Dual-color stained Bis-Tris native gel demonstrating how different seeds, including SNAP–RIP2–CARD (1 or 5 μM), SNAP–NOD1–CARD (10 μM), and SNAP–NOD2–CARD (10 μM), colored in green, triggered the induced oligomerization process of SNAP–RIP2–CARD (10 μM) monomers, colored in red. Monomer channel was shown in the lower panel in black and white. d Negative stain EM images showed that 10 μM RIP2–CARD proteins were induced to form filaments by 10 μM NOD1–CARD or 10 μM NOD2–CARD. Scale bar: 100 nm. e Cartoon illustration of NF-κB promoter luciferase assay with SNAP–RIP2–CARD protein seed directly electroporated into the cells. f NF-κB was activated by intracellular recombinant RIP2–CARD seeds. SNAP–RIP2–CARD seeds, SNAP–RIP2–CARD (K513E) seeds, and MAVS–CARD–SNAP were electroporated into HEK293T cells, together with pGL4.32 NF-κB-RE vector and CMV-Renilla vectors. Only oligomeric RIP2 protein constructs led to activation of NF-κB, but not the K513E, the monomeric form of RIP2. MAVS–CARD–SNAP was used as a strong positive control. The same amount of proteins were transfected in each sample. BG-Alexa 488 dye stained SDS-PAGE conferring that the similar level of recombinant seeds were catapulted into the reporter cells. Error bar represents standard deviation values of three independent repeats
