Fig. 4. TRAF3 promotes STAT6 ubiquitination and enhances IL-4 induced STAT6 transcriptional activity.

A HEK293 cells were co-transfected with His-STAT6, Flag-TRAF3 and HA-Ub expression plasmids. Ubiquitination assay of STAT6 was performed using anti-His antibody for IP and anti-Ub antibody for immunoblotting of ubiquitinated STAT6. B Schematic representation of the mouse STAT6 protein, showing the potential TRAF3-related ubiquinated lysine (K) residues (K129 and K450) highlighted in red. C Amino acid sequence alignment of STAT6 among the indicated species, showing K129 and K450 highlighted in red. D HEK293 cells were transfected with indicated plasmids and luciferase assay was performed to determine transcriptional activity of STAT6 or indicated STAT6 mutants (K129R and K450R) with or without TRAF3 expression. Cells were stimulated with IL-4 (10 ng/ml) for 24 h before harvest. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. One-way ANOVA, *p < 0.05, **p < 0.01, n = 3 for each group. E His-STAT6 WT or His-STAT6 K450R were transfected into HEK293 cells together with Flag-TRAF3 and HA-Ub K63. Cells were treated with (+) or without (−) IL-4 (10 ng/ml) for 2 h. Nuclear and cytosol fractions were extracted and p-STAT6 levels were detected by immunoblotting. LaminB shows nuclear fraction and Tubulin shows cytosol fraction.