Fig. 6
From: The zinc-finger protein ZFYVE1 modulates TLR3-mediated signaling by facilitating TLR3 ligand binding
Poly(I:C) stimulation causes the partial colocalization of ZFYVE1 with endosomes. a Subcellular localization of ZFYVE1 and its mutants. HEK293 cells (1 × 105) were transfected with HA-tagged ZFYVE1, ZFYVE1 (W543A) or ZFYVE1 (C654/770S) plus YFP-EEA1 (early endosomal marker). Twenty hours later, the cells were or were not treated with poly(I:C) (50 μg/mL) for 20 min. The cells were then fixed with 4% paraformaldehyde and stained with anti-ZFYVE1 antibody before confocal microscopy. b Subcellular localizations of endogenous ZFYVE1. 293-TLR3 cells (2 × 107) were or were not treated with poly(I:C) (100 μg/mL) for 30 min. Cellular fractions were analyzed by immunoblotting with the indicated antibodies. c The C-terminal FYVE domains of ZFYVE1 are located at the endosomal lumen. 293-TLR3 cells stably expressing HA-ZFYVE1-Flag (3 × 108) were treated with poly(I:C) (100 μg/mL) for 30 min. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (2 μg/mL), or treated with proteinase K plus 0.2% Triton X-100 for 15 min at 55 °C. The samples were then analyzed by immunoblotting with the indicated antibodies. d Effects of ZFYVE1 and its mutants on poly(I:C)-induced activation of the IFN-β promoter. 293-TLR3 cells (1 × 105) were transfected with the IFN-β reporter and the indicated plasmids for 24 h and then were or were not treated with poly(I:C) (20 μg/mL) for 6 h before luciferase assays. Expression of transfected ZFYVE1 and its mutants was detected by immunoblotting analysis. e Effects of reconstitution with ZFYVE1, ZFYVE1 (W543A) or ZFYVE1 (C654/770S) on the poly(I:C)-induced transcription of downstream antiviral genes. Zfyve1−/− MLFs were reconstituted with murine ZFYVE1, ZFYVE1 (W543A), or ZFYVE1 (C654/770) by lentiviral-mediated gene transfer. The reconstituted MLFs were or were not treated with poly(I:C) for the indicated times before qPCR analysis. f Zfyve1 deficiency inhibits the poly(I:C)-induced tyrosine phosphorylation of TLR3. Zfyve1-deficient MLFs stably expressing Tlr3-Flag were reconstituted with vector or Zfyve1, and the cells (4 × 107) were or were not treated with poly(I:C) (100 μg/mL) for the indicated times before coimmunoprecipitation and immunoblotting analysis