Fig. 7.

ZFYVE1 facilitates the binding of TLR3 to poly(I:C). a ZFYVE1 binds to poly(I:C) but not HSV120. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids. Eighteen hours later, cell lysates were incubated with biotinylated poly(I:C) or biotinylated HSV120 and streptavidin sepharose beads for in vitro pulldown assays. The bound proteins were analyzed by immunoblotting with anti-Flag or anti-HA antibody. b ZFYVE1 binds to poly(I:C) through its FYVE domains. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids. Eighteen hours later, cells were collected for in vitro pulldown assays performed as described in a. c Wild-type, but not W543A or C654/770S ZFYVE1 enhances the binding of poly(I:C) to TLR3. HEK293 cells (2 × 10⁶) were transfected with the indicated plasmids. Eighteen hours later, cells were collected for in vitro pulldown assays performed as described in a. d Zfyve1 deficiency inhibits the binding of poly(I:C) to TLR3. Zfyve1-deficient MLFs stably expressing Tlr3-Flag were reconstituted with vector or murine ZFYVE1, and the cell lysates (2 × 10⁸ each) were then incubated with biotinylated poly(I:C) and streptavidin sepharose beads for in vitro pulldown assays. The bound proteins were then analyzed by immunoblotting with anti-Flag antibody. e ZFYVE1 increases the binding affinity of poly(I:C) to TLR3. Binding affinities between the indicated recombinant proteins or between the indicated recombinant proteins and poly(I:C) were measured by MST. The purified recombinant proteins were stained with coomassie blue (right panel)