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. 2012 Nov 30;7(11):e50637. doi: 10.1371/journal.pone.0050637

Figure 3. Mapping of FOG-2 SUMOylation.

Figure 3

(A) A series of FOG-2 deletion fragments (509-1121, 729-1151, 881-1092) were transfected into COS-7 cells together with expression vectors for HA-SUMO-1 or GFP-SUMO-1 as indicated in the figure. Mutations in fragments 279-1151 and 881-1092 are indicated in the figure. All FOG-2 fragments were SUMOylated. Mutation of K915 and K955 in the 881-1092 fragment caused the disappearance of the previous SUMOylation band (lane 4, lower panel), indicating that K955 is a site for SUMO modification in FOG-2. (B) FOG-2 wt and single, triple and quadruple mutants were co-expressed with GFP-SUMO-1 (lanes 3 to 9). The SUMOylated FOG-2 species are indicated by black dots in the blot and by arrowheads (upper panel). Mutation of K955 (lane 7) abolished the fourth SUMOylation band, indicating that, apart from K955, FOG-2 possesses 3 additional SUMO acceptor sites. Mutation of the other 3 sites (K324/471/915) results in a single SUMOylation band corresponding to K955 (lane 8). Mutation of K324/471/915/955R led to the abolition of FOG-2 SUMOylation (lane 9). The expression of GFP-SUMO-1 and the total SUMOylation levels in the cell extracts are shown in the middle and lower panels, respectively. IB, immunoblot.