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. 2015 Dec 1;16(12):1215–1227. doi: 10.1038/ni.3279

Figure 7. DTX3L mediates immunoproteasomal degradation of viral 3C protease.

Figure 7

(a) Immunoblot analysis of EMCV 3C in various U3A (left) or U3A-STAT1 (right) cell lines (above lanes) at 6 h after infection with EMCV (MOI, 1). (b) Immunoblot analysis of EMCV 3C and PSMB8 in various U3A cell lines (above lanes) left untreated (−) or treated (+) for 1 or 2 h (below blot) with the PSMB8-specific inhibitor ONX-0914 (0.1 μM) and then infected for 6 h with EMCV (MOI, 1). (c) EMCV RNA in cells as in a. (d) Immunoblot analysis of various proteins (left margin) in various U3A-STAT1 cell lines (above lanes) treated for 6 h with IFN-β (10 U/ml) or not (below blot) and then infected for 18 h with EMCV (MOI, 1). (e) Immunoblot analysis of EMCV 3C and other proteins (left margin) in HEK293T cells transfected for 36 h with various vectors (above lanes) alone (−) or together with (+) vectors encoding histidine- and V5-tagged EMCV 3C (His-V5-EMCV-3C) plus histidine-tagged ubiquitin (His-Ub) and then treated for 14 h with MG-132 (20 μM) (+MG-132) or not (−MG-132) (below blots). (f) Immunoblot analysis of EMCV 3C in human tracheal epithelial cells infected with EMCV at an MOI of 1 or 10 (top) and then, at 32 h later, treated for 12 h with MG-132 (20 μM) (+) or not (−). (g) EMCV 3Dpol RNA in cells as in f. (h) Immunoblot analysis of primary cultures of human tracheal epithelial cells with or without treatment for 24 h with interferon (above lanes), showing the co-immunoprecipitation of endogenous STAT1 with PARP9-DTX3L (top), and input controls (below). Data are representative of three independent experiments (mean and s.e.m. in c,g).