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. 2022 May 19;5:483. doi: 10.1038/s42003-022-03427-4

Fig. 4. The C-terminal sequence of ORF6 disrupts the nuclear localization of STAT1.

Fig. 4

a The bacterially purified STAT1 recombinant protein was incubated with either GST-GFP or GST-GFP-ORF6 immobilized on glutathione Sepharose beads (GST-beads) for 1 h, and the pulled-down protein was collected (indicated as PD). STAT1 was detected with anti-STAT1 antibody (WB). The bottom panel represents the proteins bound to the GST-beads and stained with Coomassie Brilliant Blue (CBB). The right lane was loaded with the STAT1 protein (1/15,000 dilution of the reaction) as an input protein. Values are kDa. b The STAT1 recombinant protein was incubated with either GST-GFP or GST-GFP fused with the C-terminal peptide of ORF6 wild type (49-61 amino acids; GST-M0-GFP) immobilized on GST-beads for 1 h, and then a pulled-down protein was collected (PD). STAT1 was detected using anti-STAT1 antibody (WB). The bottom panel represents the proteins bound to the beads and stained with CBB. The right lane loaded with the STAT1 protein (1/10,000 dilution of the reaction) as an input protein. Values are kDa. c The STAT1 recombinant protein was incubated with either GST-GFP, GST-GFP-ORF6 wild type (WT), ore GST-GFP-ORF6 C-terminal deletion mutant lacking a.a. 49–61 (ΔC) immobilized on GST-beads for 1 h, and then a pulled-down protein was collected (PD). STAT1 was detected using anti-STAT1 antibody (WB). The bottom panel represents the proteins bound to the beads and stained with CBB. The right lane loaded with the STAT1 protein (1/15,000 dilution of the reaction) as an input protein. Values are kDa. d Lysates from HeLa cells stimulated with IFN-γ were incubated with either GST-GFP or GST-GFP-ORF6 immobilized on GST-beads for 1 h, and the pulled-down proteins were collected (PD). PY-STAT1 was detected with a specific antibody (WB). The bottom panel represents the proteins bound to the beads and stained with CBB. The input was a 1/500 dilution of cell lysates used for the reaction. Values are kDa. e Lysates from HeLa cells stimulated with IFN-γ were incubated with GST-GFP, GST-M0-GFP, GST-M1-GFP, GST-M2-GFP, or GST-M3-GFP, which were immobilized on GST-beads for 1 h, and the pulled-down proteins were collected (PD). PY-STAT1 was detected with a specific antibody (WB). The bottom panel represents the proteins bound to the beads and stained with CBB. The input was a 1/1,000 dilution of cell lysates used for the reaction. f, g Huh7 cells were microinjected with either GST-GFP, GST-M0-GFP, GST-M1-GFP, GST-M2-GFP, or GST-M3-GFP proteins (green), as well as a Fluorescence-conjugated antibody (red) as an injection marker, into the cytoplasm. The injected cells were incubated f or treated without IFN-γ g for 30 min, and PY-STAT1 (magenta) was detected using specific antibodies. DAPI was used for DNA staining (blue). Scale bars: 30 μm.