Figure 5.
Dppa2 and Dppa4 Are Major Endogenous SUMO Substrates in ESCs
(A) Top 20 substrates differentially modified by SUMO2/3 in MEFs and ESCs. Pluripotency proteins are highlighted in red. SUMO2/3 targets are ranked with the top hits at the bottom for MEFs and at the top for ESCs. Color gradient is for didactic purposes and does not correlate to ratio of change.
(B) Correlation between protein abundance (x axis) and the SUMO2/3 abundance for the corresponding proteins (y axis) in ESCs. Pluripotency factors are highlighted in red. The black dotted line corresponds to the regression line for all SUMO substrates and the red one to pluripotency factors only.
(C) Immunoblots for Dppa2 and Dppa4 in ESCs after 2 day continuous treatment with DMSO or 2 μM ML-792. The star indicates a non-specific band. Representative example, n = 3. Two independent blots are shown. Ponceau staining was used as a loading control.
(D) Schematic representation of Dppa2 and Dppa4 depicting SUMOylation sites identified by mass spectrometry. The percentage indicates the contribution of each lysine to the total SUMOylation of the substrate. In red, the main lysine residues, which were mutated to arginine residues. SAP (SAF-A/B, Acinus, and PIAS) domain is indicated.
(E) SUMOylation of Dppa2 WT or Mut (left) and Dppa4 WT or Mut (right) in vivo. Lysates from HeLa cells transfected with Dppa2 (WT or Mut) or Dppa4 (WT or Mut) together with Ubc9 and HA-SUMO2 and immunoblotted with indicated antibodies
(F) Immunoblot of Dppa2 (left) in HeLa cells overexpressing 6His-SUMO2 together with Dppa2 wild-type (WT) or Dppa2 mutant (Mut). Immunoblot of Dppa4 (right) in HeLa cells overexpressing 6His-SUMO2 together with Dppa4 wild-type (WT) or Dppa4 mutant (Mut). Forty-eight hours post-transfection, cells were harvested, an aliquot of the lysates was directly analyzed (input), and the remaining extracts were used for Ni2+ affinity chromatography to purify 6His-SUMO2 conjugates (Ni-NTA).