Fig 7.

TRIM22 induces ubiquitination of NP. (A) Flag-NP was coexpressed with His-Ubi and increasing amounts of TRIM22 in 293T cells. Forty-eight h after transfection, the cells were treated with 10 μM MG132 for 3 h. Subsequently, WCE were prepared from a 10% fraction of the cells and analyzed by Western blotting using anti-Flag (upper), anti-TRIM22 (middle), and anti-tubulin (lower) antibodies. (B) The remaining 90% of the cells were lysed in 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. WCE from Flag-NP-expressing 293T cells (WCE Flag-NP) was loaded on the gel for size comparison.