Both the UBL1 and HVR domains of Nsp3 are required for recruitment of FXRs, related to Figs. 5 and 6. (A) The schematic shows the domain organization of the truncated forms of Nsp3. (B) In a Myc-Trap assay, mCherry-Nsp3(UBL1+C) or mCherry-Nsp3(HVR+C) is marginally precipitated by Myc-FXR1. Myc-FXR1 pulls down similar levels of mCherry-Nsp3(UBL1+HVR+C) as WT mCherry-Nsp3. (C and D) Cells coexpressing mCherry-Nsp3(UBL1+C) or mCherry-Nsp3(HVR+C) with Nsp4-GFP contain numerous tiny Nsp3/4⁺ puncta that do not colocalize with endogenous FXR1 (C). FXR1⁺ puncta colocalize well with mCherry-Nsp3(UBL1+HVR+C)/Nsp4-GFP double-positive dots (D). Bars: 5 μm; insets, 2 μm. (E) The UBL1 or HVR domain of Nsp3 enters into FXR1 droplets at a lower level than Nsp3N. A fragment containing both HVR and UBL1 domains strongly segregates into FXR1 phases. Bars: 5 μm. (F) The schematic shows full-length FXR1 and mutants with deletions of different domains. (G–M) Full-length FXR1 (G), GFP-FXR1(ΔAL1ΔAL2) (H), GFP-FXR1(KH1-C) (I), GFP-FXR1(KH2-C) (J), and GFP-FXR1 (ΔKH2) (L) all show similar colocalization with Nsp3/4⁺ puncta, while GFP-FXR1(IDR1-C) (K) or GFP-FXR1 (ΔNIR) (M) fails to be recruited to Nsp3/4⁺ foci. Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm. (N) Full-length FXR1, GFP-FXR1(ΔAL1ΔAL2), GFP-FXR1(KH1-C), GFP-FXR1(KH2-C), GFP-FXR1(IDR1-C), GFP-FXR1 (ΔKH2), and GFP-FXR1(ΔNIR) express well in cells. Source data are available for this figure: SourceData FS4.