Figure S2.
Analyses of ORF52 phase separation. (A) Plasmids expressing different tegument proteins were individually transfected into COS-7 cells. At 24 h post transfection, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100. Indirect immunofluorescence analysis was carried out using an antibody against HA-tag or Flag-tag. Bar: 5 μm. (B) Purity of bacterially expressed ORF52, analyzed by Coomassie blue staining. (C) Phase separation diagram of ORF52 at the indicated concentrations. ORF52 with the indicated concentrations were incubated in phase separation assay buffer with different concentrations of NaCl and visualized by confocal microscopy. P.S, phase separation. (D) Expression levels of ORF52 in 293T cells after MHV-68 infection (MOI = 3) at different hours post infection, as examined by Western blotting. con1-3: Bacterially expressed and purified ORF52 proteins were loaded at the indicated amount to draw standard curve. (E) Phase separation assay of ORF52 with v-tRNA was performed in physiological buffer. 10 μM ORF52 protein (3% Alexa 488-labeled) was mixed with 100 ng/μl v-tRNA in 96-well plates coated with 20 mg/ml BSA. Mixtures were incubated and images were captured by confocal microscopy. Bar: 10 μm. (F) Phase separation assay of ORF52 with total RNA extracted from infected or uninfected cells was performed in physiological buffer. 10 μM ORF52 protein was mixed with 100 ng/μl total RNA in 96-well plates coated with 20 mg/ml BSA. Mixtures were incubated and images were captured by confocal microscopy. Bar: 10 μm. Source data are available for this figure: SourceData FS2.

Analyses of ORF52 phase separation. (A) Plasmids expressing different tegument proteins were individually transfected into COS-7 cells. At 24 h post transfection, the cells were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100. Indirect immunofluorescence analysis was carried out using an antibody against HA-tag or Flag-tag. Bar: 5 μm. (B) Purity of bacterially expressed ORF52, analyzed by Coomassie blue staining. (C) Phase separation diagram of ORF52 at the indicated concentrations. ORF52 with the indicated concentrations were incubated in phase separation assay buffer with different concentrations of NaCl and visualized by confocal microscopy. P.S, phase separation. (D) Expression levels of ORF52 in 293T cells after MHV-68 infection (MOI = 3) at different hours post infection, as examined by Western blotting. con1-3: Bacterially expressed and purified ORF52 proteins were loaded at the indicated amount to draw standard curve. (E) Phase separation assay of ORF52 with v-tRNA was performed in physiological buffer. 10 μM ORF52 protein (3% Alexa 488-labeled) was mixed with 100 ng/μl v-tRNA in 96-well plates coated with 20 mg/ml BSA. Mixtures were incubated and images were captured by confocal microscopy. Bar: 10 μm. (F) Phase separation assay of ORF52 with total RNA extracted from infected or uninfected cells was performed in physiological buffer. 10 μM ORF52 protein was mixed with 100 ng/μl total RNA in 96-well plates coated with 20 mg/ml BSA. Mixtures were incubated and images were captured by confocal microscopy. Bar: 10 μm. Source data are available for this figure: SourceData FS2.

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