Fig. 4.

TRAF4 mediates AR ubiquitination at the K913 residue. (A) TRAF4 mainly targets AR LBD for ubiquitination. Upper, schematic representation of WT, splicing variant (V7) or deletion mutants of AR. Bottom (from the top to bottom), the ubiquitination levels of flag-AR WT or its mutant in cells (Shown is a western blot analysis using an HA-specific Ab to detect HA-ubiquitinated AR); the level of flag-AR or its mutants following an IP with a flag Ab; the levels of TRAF4 from the inputs and β-actin as a loading control of the input. (B) Deletion of the last 53aa in AR abolished TRAF4-mediated ubiquitination. 293T cells were transiently transfected with flag-AR WT or the deletion mutant (1-867), TRAF4 WT, ΔRING, or vector control, together with HA-ubiquitin, V5-TRAF4. An IP was carried out using a flag Ab followed by western blot analysis. Bottom, the amino acid sequence of the last 53aa, which contains 4 lysine residues. (C) K913R mutation abolished TRAF4-mediated AR ubiquitination.