Figure 1: Identification of candidate SUMO sites on MeCP2.
(a) In vitro SUMOylation assay showing MeCP2 SUMOylation by PIAS1. Purified GST-E1, His-E2, His-PIAS1, GST-MeCP2 and GST-SENP1 proteins were added to the reaction for this assay. (b) V5-MeCP2 and Myc-SUMO1 plasmids were co-transfected with different amounts of Flag-PIAS1 plasmid to HEK293T cells confirming MeCP2 SUMOylation by PIAS1. (c) SUMO2.0 Software prediction of candidate SUMO acceptors on MeCP2. The ‘K’ letter indicated by the arrow represents the candidate SUMO sites. (d) Flag-PIAS1 and Myc-SUMO1 (or Myc-SUMO1ΔGG) plasmids were co-transfected with V5-MeCP2WT or different V5-MeCP2 lysine mutant plasmids to HEK293T cells. MeCP2 SUMOylation was examined by immunoblotting using anti-V5 antibody. The quantified result is shown in the lower panel (n=2 each group; F9,10=240.59, #P<0.001; q=12.73, #P<0.001 comparing lane 3 versus lane 9; q=21.1, #P<0.001 comparing lane 3 versus lane 10; q=8.37, **P<0.01 comparing lane 9 versus lane 10, one-way ANOVA followed by Newman–Keul post hoc multiple comparisons). (e) V5-MeCP2 or V5-MeCP2K412R plasmid was co-transfected with Myc-SUMO1 and Flag-PIAS1 plasmids to HEK293T cells. The cell lysate was immunoprecipitated with anit-Myc antibody and immunoblotted with anti-V5 antibody for confirmation of MeCP2 SUMOylation at Lys-412. The arrow indicates MeCP2 SUMOylation at Lys-412. All experiments are in two repeats. Data are expressed as mean±s.e.m.
