Fig. 6

ENO-RPSA-VIM forms co-condensates on the cell membrane. A and B After the corresponding time of SS2 or ENO (20 μg/mL) protein stimulation for HCMEC/D3 cells, cell lysates were used for western blot analysis. Quantification was performed by Image J (n = 3 biologically independent samples, mean ± SD). C After SS2 infection of HCMEC/D3 cells for 1 h, the localization of RPSA and VIM on the cell surface was observed by immunofluorescence staining using the antibodies against RPSA and VIM. Representative confocal images are shown (scale bar = 20 μm) and magnified (scale bar = 10 μm). Pearson and Overlap Coefficients were quantitatively analyzed by Image J (n = 5 fields in each group, a field contains about 8 cells, mean ± SD). D—F HEK-293 T cells were co-transfected with the indicated plasmids co-expressing GFP-RPSA and FLAG-VIM proteins (GFP-RPSA and FLAG, GFP and FLAG-VIM). D After 36 h, cell lysates were immunoprecipitated with the antibody against GFP or FLAG, followed by western blot analysis using the indicated antibodies against FLAG or GFP. E and F After 12 h, ENO (20 μg/mL) protein was added to the transfected cells for stimulation. After stimulation for the indicated times, cells were treated with (E) or without (F) 1,6-Hex. Cell lysates were immunoprecipitated with antibody against GFP, followed by western blot analysis using the antibody against FLAG. Quantification was performed by Image J as mean ± SD (n = 3 biologically independent samples). G Multiples fluorescence immunohistochemistry analyses of the co-localization of RPSA and VIM proteins from brain tissue of healthy and SS2-infected piglets. Representative stained tissue slices were shown (scale bar = 50 μm). A-C, E and F * for P < 0.05, ** for P < 0.01, *** for P < 0.001; (A, B and E) one-way ANOVA with Tukey’s test, (C and F) two-tailed unpaired Student’s t-test