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. Author manuscript; available in PMC: 2017 Apr 18.
Published in final edited form as: Cell Rep. 2017 Mar 28;18(13):3167–3177. doi: 10.1016/j.celrep.2017.03.003

Figure 1. GSK3 phosphorylates Olig2 at S10.

Figure 1

(A and B) Inhibition of GSK3 results in reduced Olig2 phosphorylation. Mouse NPCs and BT145 human glioma cells treated with GSK3 inhibitors, CHIR99021(CHIR), SB216763(SB) or LiCl for 4 hours show a decrease in P-Olig2 level, in comparison to control cells that were treated with either DMSO or NaCl. 5 μm CHIR99021, 10 μm SB216763, 10 mM of NaCl and 10 mM LiCl were used. Relative P-Olig2/Olig2 levels were quantified and compared between control and inhibitor-treated group. Data were analyzed by t-test and are represented as mean ± SEM. *p<0.05, **p<0.01, n=3. (C) A schematic diagram shows generation of mouse GSK3α- and β-knockout NPC lines. Control adeno or adeno-cre virus was introduced to generate GSK3α- or GSK3β-knockout NPC lines. (D and E) GSK3α and β function redundantly to phosphorylate Olig2. P-Olig2 levels were examined in GSK3α−/+, GSK3α −/−, and GSK3α−/+GSK3β−/− NPCs. Relative P-Olig2/Olig2 were quantified and compared between different groups. Data were analyzed by t-test and are represented as mean ± SEM. *p<0.05, n=3. (F) GSK3 consensus motif fits the S10 site upon phosphorylation at S14. S/T (highlighted with red), the kinase’s target Serine/Threonine residue; X, any amino acid; pS, phosphorylated serine. (G) in vitro kinase assay shows that GSK3 phosphorylates Olig2 N-terminal peptide, however, it requires a priming phosphorylation at S14. Synthetic Olig2 N-terminal peptides (a.a.1–18) were used, and both unphosphorylated Olig2 N-terminal peptide (unPO4) and phosphorylated peptide at S14 (pS14) were tested. Reactions without peptides (none) served as negative control. Data were analyzed by t-test and are represented as mean ± SEM. n=3; **p<0.01. (H) Mapping the GSK3 phosphorylation sites by mass spectrometry analysis. in vitro kinase reactions were analyzed by MALDI-MS and −MS/MS. The doubly phosphorylated peptide at S14 and S10 is indicated by pS14/S10.