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. 2019 Jun 21;17(6):e3000324. doi: 10.1371/journal.pbio.3000324

Fig 5. The sumoylation of DPPA2 by PIAS4 depends on K31 and K108 sites.

Fig 5

(A) Western blotting analysis of Flag IP samples in ESCs overexpressing Dppa2–3XFlag and 3XHA–Sumo2 and treated with control or Pias4 siRNAs. Quantification of relative sumoylation level is shown below. Data were normalized to siNC-treated ESCs overexpressing Dppa2–3XFlag and 3XHA–Sumo2. Shown are mean ± SD, n = 3. The p-value was calculated by two-tailed Student’s t test. (B) Western blotting analysis of FLAG–DPPA2 and 3XHA–SUMO2 in ESCs transfected with siNCs or Pias4. Left, representative images of gel blots; right, quantification of FLAG–DPPA2 and 3XHA–SUMO2. Data were normalized to GAPDH and then to Flag–Dppa2/3XHA–Sumo2 overexpressing ESCs treated with siNC. Shown are mean ± SD, n = 3. The p-value was calculated by two-tailed Student’s t test. (C) Western blotting analysis of Flag IP samples in HEK293T cells transfected with Dppa2–3XFlag and 3XHA–Sumo2 with or without overexpression of Pias4. (D) Table of the predicted SUMOylation sites using in silico prediction programs SUMOplot and GPS-SUMO in DPPA2. Scores ware based on two criteria: direct amino acid match to SUMO consensus motif (ψKXE) or substitution of the consensus amino acid residues with amino acid residues exhibiting similar hydrophobicity. (E) Western blotting analysis of Flag IP samples in HEK293T cells transfected with flag-tagged lysine mutant, 3XHA-Sumo2, and Pias4. Quantification for relative sumoylation level is shown below. Data were normalized to HEK293T cells transfected with flag-tagged WT Dppa2, 3XHA-Sumo2, and Pias4. Shown are mean ± SD, n = 3. The p-value was calculated by one-way ANOVA followed by two-tailed Dunnett's test. Source data for A, B, and E can be found in the supplemental data file (S1 Data). BFP, blue fluorescent protein; Dppa2, developmental pluripotency associated 2; ESC, embryonic stem cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HA, hemagglutinin; HEK293T, human embryonic kidney 293T; IP, immunoprecipitation; PIAS4, protein inhibitor of activated STAT 4; siNC, siRNA against negative control; siRNA, small interfering RNA; SUMO, small ubiquitin-like modifier; WT, wild type.