Figure 2

PRRSV Nsp2 interacts with TBK1 and induces its degradation via the lysosomal pathway. A HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and Flag-TBK1 or HA/Flag-tagged empty vector for 36 h, followed by co-IP with anti-HA/Flag magnetic beads and IB analyses with anti-HA/Flag antibodies. B HeLa cells were transfected with the plasmids encoding Nsp2-HA and Flag-TBK1 for 24 h. In parallel, HeLa cells were transfected with the plasmid encoding Nsp2-HA and Flag-tagged empty vector, or Flag-TBK1 and HA-tagged empty vector. Flag-TBK1 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. C 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-TBK1 antibodies. D MARC-145 cells were infected with PRRSV at an MOI of 0.1 for 24 h. The fluorescent signals were observed with confocal microscopy (scale bars = 10 µm). The co-localisation was assessed by determining the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. E HEK-293 T cells were co-transfected with the plasmids encoding Flag-TBK1 and Nsp2-HA or Nsp2-mutant-HA. At 24 h post-transfection, the cell lysates were collected to analyse the TBK1 protein level with IB. F HEK-293 T cells were transfected with the plasmids encoding Nsp2-HA and Flag-TBK1 for 6 h. 3-MA, CQ, MG132, or DMSO was added and the samples were collected after 24 h for IB analyses with anti-HA and anti-Flag antibodies.