Confirmation of FOG-2 SUMOylation sites.
(A) COS-7 cells were co-transfected with GFP or GFP-SUMO-1 and either wt FOG-2, FOG-2 triple SUMOylation site mutant (K324/471/915R) or FOG-2 quadruple mutant (K324/471/915/955R). Immuno-precipitation experiments were performed in cell extracts using magnetic beads coated with an anti-GFP antibody. Immuno-precipitated complexes and cell lysates (input) were resolved by SDS-PAGE and blotted with anti-FOG-2, anti-GFP or anti-SUMO-1 antibodies. Immuno-precipitated FOG-2 SUMOylated by GFP-SUMO-1 is observed in lane 2, upper panel. A single SUMOylated band is seen in the triple mutant (lane 3, upper panel), while the FOG-2 K324/471/915/955R mutant is not SUMOylated (lane 4, upper panel). Protein input (5%) in shown in lanes 5 to 8. Expression of GFP alone (lanes 1 and 5), GFP-SUMO-1 (lanes 2–4 and 6–8) and total SUMOylation are shown in the middle and lower panels. Asterisks indicate non-specific bands detected by the FOG-2 antibody (this non-specific band was enriched by the immuno-precipitation - upper panel, lanes 2 to 4 - indicating that this cross-reacting species is also a SUMOylated protein). (B) The alignment of FOG-2 sequences from human to platypus (Ornithorhynchus anatinus) shows conservation of the four SUMOylation sites. Asterisks indicate non-specific bands detected by the FOG-2 antibody. IB, immunoblot; IP, immuno-precipitation.