Fig. 3.

O-GlcNAc of mouse Gli2 occurs at S355. (A) Coimmunoprecipitation of OGT and Gli2 from the cerebellum of WT mice at P7. (B) O-GlcNAc of Gli2 on serine 355 in vivo. Gli2 was purified from mice cerebellum and analyzed by MS. (C) Cross-species sequence alignment of Gli2 revealed conserved O-GlcNAc sites at S273, S355, S361, T362, T791, and S844, respectively. (D and E) Representative immunoblot (D) and quantification (E) of the O-GlcNAcylation levels of immunoprecipitated WT and mutant Gli2 constructs. HEK293T cells were transfected for 48 h with Myc-tagged WT or the indicated mutant Gli2 construct (n = 3). One-way ANOVA and Dunnett’s multiple comparisons test. (F) Luciferase readout in NIH 3T3 cells treated with 0.1 μM SAG for 36 h and cotransfected for 48 h with pCMV6-vector (pCMV6), WT Gli2, or Gli2 mutants S273A, S355A, S361/T362A, T791A, S844A with Bcl2 promoter luciferase reporter plasmid and Renilla luciferase reporter plasmid pRL-TK as internal reference (n = 3). One-way ANOVA and Dunnett’s multiple comparisons test. (G) Relative luciferase activity in HEK293T cells cotransfected for 48 h with pCMV6-vector (pCMV6), WT Gli2 or Gli2 S355A mutant, the Bcl2 promoter luciferase reporter plasmid and Renilla luciferase reporter plasmid pRL-TK, with pEGFP-C1 vector (pEGFP-C1), pEGFP-C1-OGT (OGT), or pEGFP-C1-OGT^Mut (H498A/H558A double mutant, termed as OGT^Mut), respectively (n = 3). One-way ANOVA and Dunnett’s multiple comparisons test. (H) Relative luciferase activity in HEK293T cells cotransfected for 48 h with pCMV6-vector (pCMV6), WT Gli2 or Gli2 S355A mutant, the Bcl2 promoter luciferase reporter plasmid and Renilla luciferase reporter plasmid pRL-TK. Transfected cells were treated with OGT inhibitor OSMI-1 (5 μM), OGA inhibitor TM-G (80 μM), and DMSO as control for 36 h (n = 3). One-way ANOVA and Dunnett’s multiple comparisons test. All data represent mean ± SEM *P < 0.05, **P < 0.01. See also SI Appendix, Figs. S7–S10.