Figure 3

PRRSV Nsp2 interacts with HSPA8. A HEK-293 T cells were transfected with the plasmid expressing HA or Nsp2-HA. The proteins were immunoprecipitated in cell lysates using an anti-HA antibody, separated by 12% SDS-PAGE, and stained with silver. The red arrow indicates the significantly different immunoprecipitated protein band. The black arrow marks Nsp2-HA. The panel on the right shows the tandem MS analysis of HSPA8 peptides. B HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and HSPA8-myc or HA/myc-tagged empty vector for 36 h, followed by co-IP with anti-HA or anti-myc magnetic beads, and IB analyses with anti-HA and anti-myc antibodies. C HeLa cells were transfected with the plasmids encoding Nsp2-HA and HSPA8-myc for 24 h. In parallel, HeLa cells were transfected with the plasmid encoding Nsp2-HA and myc-tagged empty vector, or HSPA8-myc and HA-tagged empty vector. HSPA8-myc and Nsp2-HA were visualised with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. The fluorescent signals were observed with confocal microscopy (scale bars = 10 µm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. D MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. They were then analysed via endogenous IP using protein A/G magnetic beads pre-incubated with anti-Nsp2 pAbs, and IB with anti-Nsp2 and anti-HSPA8 antibodies. E MARC-145 cells were infected with PRRSV at an MOI of 1 for 24 h. The fluorescent signals were observed with confocal microscopy (scale bars = 10 µm). The co-localisation was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software.