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. 2018 Apr;517:164–176. doi: 10.1016/j.virol.2017.12.017

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© 2017 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Fig. 2 — The de-ubiquitination and de-ISGylation activities of PRRSV nsp2 related proteins. (A) Effect of nsp2 related protein expression on ubiquitin conjugation. HEK-293T cells were co-transfected with plasmid DNAs expressing HA-Ub and FLAG-tagged nsp2, nsp2TF, nsp2N, PLP2 or their catalytic site mutants (nsp2-C/H>A, nsp2TF-C/H>A, nsp2N-C/H>A, PLP2-C/H>A). HA-Ub conjugated cellular proteins were visualized by western blot analysis using anti-HA mAb. The expression of FLAG-tagged nsp2 related proteins was detected by anti-FLAG M2 mAb. (B) HEK-293T cells were co-transfected with plasmid DNAs expressing ISG15 and its conjugation enzymes E1/E2/E3, FLAG-tagged nsp2, nsp2TF, nsp2N, PLP2 or their catalytic site mutants (nsp2-C/H>A, nsp2TF-C/H>A, nsp2N-C/H>A, PLP2-C/H>A). ISG15-conjugated cellular proteins were detected by ISG15 specific mAb in western blot analysis. The expression of FLAG-tagged nsp2 related proteins was detected by anti-FLAG M2 mAb. For both assays, the empty p3xFLAG vector plasmid was included as a control, while expression of housekeeping gene GAPDH was detected as a loading control.