Fig. 4.

K48- and K63-linked ubiquitination of cellular TRIM21 requires Ube2W and Ube2N. (A–C) TE671 expressing a scrambled shRNA (Scr), Ube2N-specific shRNA, or Ube2W-specific shRNA (B) and the indicated combinations of 6xHis-TRIM21 and HA-Ub. Cell lysates were incubated with Ni²⁺ affinity resin, and input and pull-down (PD) fractions were resolved by LDS-PAGE and detected by IB with antibodies against total Ub, Ube2N, Ub-⁴⁸Ub, Ub-⁶³Ub, or β-Actin (loading control). In C, 1 μg purified Ub-⁴⁸Ub or Ub-⁶³Ub was loaded to control for antibody specificity.