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. 2018 Apr 4;3(2):e00110-18. doi: 10.1128/mSphere.00110-18

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Copyright © 2018 Pagani et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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FIG 6 — pH1N1 NP is less sensitive than sH1N1 NP to TRIM22-mediated ubiquitination. (A) sH1N1 Flag-NP, pH1N1 Flag-NP, and quadruple mutant (pH1N1-K4) were cotransfected with His-Ubi under all conditions and TRIM22 in HEK293T cells. WCEs were prepared from a 10% fraction of the cells and analyzed by Western blotting using anti-Flag (upper panel), anti-TRIM22 (middle panel), and anti-β-actin (lower panel) antibodies. (B) The remaining 90% of the cells were lysed under denaturing conditions, and ubiquitinated proteins were precipitated using Ni-NTA agarose. Purified ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of Flag-NP (Ubi-NP) using anti-Flag antibodies. (C) The Flag-NP pH1N1 WT and its different K1, K2, K3, and K4 mutants were coexpressed with His-Ubi and TRIM22, in HEK293T cells. WCEs were prepared and analyzed as described for panel A. (D) Ubiquitinated forms of Flag-NP and its mutants (Ubi-NP) were analyzed as described for panel B. MW, molecular weight in thousands.