Figure 2
FOXP2 can be SUMOylated with SUMO1, 2 and 3.
(a) Schematic representation of the FOXP2-UBC9 fusion protein with an N-terminal V5 epitope tag. (b) Gel shift assay for FOXP2 SUMOylation. FOXP2-UBC9 was expressed in HEK293 cells together with a YFP-fusion of either wild-type SUMO (GG), or mutant SUMO in which the two C-terminal glycine residues required for conjugation to the target protein were mutated to alanine (AA), or with YFP alone (control). Top panel: western blot probed with anti-V5 antibody to detect FOXP2-UBC9. The 110 kDa species is unmodified FOXP2-UBC9. The 130 kDa species is FOXP2-UBC9 modified with endogenous SUMO. The 170 kDa species is FOXP2-UBC9 modified with YFP-SUMO. Middle panel: western blot probed with anti-YFP antibody. The asterisk indicates unconjugated YFP-SUMO. Higher molecular weight species are cellular proteins modified with YFP-SUMO. Bottom panel: western blot probed with anti-β-actin to confirm equal loading. (c) BRET assay for interaction between FOXP2 and SUMO. HEK293 cells were transfected with luciferase-FOXP2 (donor) and YFP-SUMO (acceptor), using either wild-type SUMO (GG) or alanine mutants (AA). The control donor protein is a nuclear-targeted luciferase. Values are mean corrected BRET ratios ± S.E.M. (n = 3).
