FIG 1.
TRP120 effector autoubiquitinates and contains a C-terminal HECT E3 ligase domain. (A) Alignment of TRP120 HECT E3 catalytic domain with other HECT-like E3 ubiquitin ligases. Conserved cysteine and leucine residues are boxed. (B) TRP120 in vitro ubiquitination assay was performed in separate reactions using recombinant protein in the presence of Ubch1, Ubch5a, and Ubch5b (left) and Ubch5c (right), with immunoblot analysis of assay products with anti-Ub. (C) Immunoblot analysis of assay products with anti-TRP120 and densitometric analysis using ImageJ software shows elevated ubiquitinated TRP120 in the presence of UbcH5b/c compared with control (bottom; *, Student's t test; P < 0.05). (D) TRP120 in vitro ubiquitination assay using recombinant protein and UbcH5b/c with or without heclin HECT E3 inhibitor, with immunoblot analysis of assay products with anti-Ub and anti-TRP120. (E) TRP120 in vitro ubiquitination assay using TRP120C520S recombinant mutant protein in the presence of E1 and UbcH5b/c, with immunoblot analysis of assay products with anti-Ub and anti-TRP120.