Figure 7.

Nsp1α inhibits K48-linked polyubiquitination and degradation of TRAIP. HEK 293T cells were co-transfected with Flag-TRAIP and Myc-nsp1α or vector supplemented 10 μM MG132 (A). At 24 h post-transfection, the cell lysates were co-immunoprecipitated with an anti-Flag and probed with Ub antibody to detect ubiquitin levels of TRAIP respectively by Western blotting. (B) 293T cells were co-transfected with Flag-TRAIP or Flag-TRAIP (K205R), HA-Ub-WT, HA-Ub-K48 (ubiquitin mutants retaining a single lysine residue), and Myc-nsp1α or vector. At 24 h post-transfection, the cell lysates were precipitated with an anti-Flag MAb and further detected by Western blotting with an anti-HA MAb and an anti-Flag. (C) The K48 polyubiquitination of TRAIP was detected in different TRAIP deletion mutants. HEK 293T cells were transfected with Myc-nsp1α (0.5 or 1.0 μg) or vector (D,E). TRAIP expression in total cellular protein and the protein stability of TRAIP in the absence of proteasome inhibitor MG132 (5 μM) was detected by Western blotting, respectively.