<p>(A) TRIM16 <i>in vivo</i> polyubiquitination assay. In HEK293 cells, HA-Ub was co-transfected with various TRIM16-GFP domain deletion expression plasmids. The protein lysate was subjected to immunoprecipitation by GFP antibody, and subjected to Western blot and probed with anti-HA antibody for Ub (right panel) and anti-GFP antibody for TRIM16 (left and middle panel). Two exposure times are shown. GFP antibodies detect both un-ubiquitinated and polyubiquitinated forms of TRIM16. Polyubiquitinated smear is present in the sample transfected with wild-type TRIM16 and shown by anti-GFP and anti-HA antibodies. (B) <i>In vitro</i> ubiquitination assay with myc-His tagged TRIM16 together with a panel of E2 enzymes, showing activity with the UbcH5 family. (C) <i>In vitro</i> ubiquitination assay with full-length TRIM16, TRIM16 domain deletion mutants or empty vector showing extensive polyubiquitination with full-length TRIM16 as detected by Western blot with anti-myc antibodies. Numbers indicate protein size in kDa. (D) Recombinant TRIM16 (Abnova) was evaluated for E3 activity in the presence of recombinant E1, UbcH5b, and HA-Ub as indicated. The capacity to catalyse auto-ubiquitination <i>in vitro</i> was observed only in the presence of ZnCl<sub>2</sub> and ATP. Western Blot (lower panel) with TRIM16 antibody showed amount of TRIM16 protein in each lane.</p
