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. 2016 Jun 17;6:28452. doi: 10.1038/srep28452

Figure 2. Effects of GlcN on O-GlcNAcylation and expression of core pluripotency factors and pluripotency of LG-ESC.

Figure 2

(a) Immunoblot of total O-GlcNAcylated proteins from LG-ESC grown ± 0.8 mM GlcN. (b) Immunoblot of NANOG, OCT4 and SOX2 from whole cell extracts (WCE) of LG-ESC, or after immunoprecipitation using nonimmune mouse IgG or O-GlcNAc antibody. (c) Immunoblot of O-GlcNAcylated proteins in whole cell extracts, or after immunoprecipitation using nonimmune rabbit IgG or antibodies against NANOG, OCT4 or SOX2. Positions of NANOG, OCT4, and SOX2 are indicated by arrows. (d) RT-PCR of Nanog, Oct4 and Sox2 mRNA normalized to rRNA from LG-ESC cultured ± 0.8 mM GlcN. (e) Immunoblot of NANOG, OCT4, SOX2, or β-ACTIN from LG-ESC. (f) Quantitation of (e). (g) RT-PCR of Nanog, Oct4, Sox2, Sox1, α-Sma, or Afp mRNA normalized to rRNA from undifferentiated (UD) or embryoid bodies (EB) LG-ESC cultured ± 0.8 mM GlcN. Experiments were repeated 2–3 times using triplicate culture dishes. Data from representative experiments are displayed as the mean ± s.e.m. (N = 3) and were analyzed by Student t-test (d,f), or one-way ANOVA followed by Tukey’s post test (g). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.