Figure 3.

Presence of O-GlcNAc stabilizes Snail1 expression by inhibiting O-phosphorylation. (A) A549 cells were grown under hyperglycaemic condition (25 mM) and cell lysates were subjected to sWGA-lectin-affinity purification and the precipitates analysed with western blot for endogenous Snail1. For control, monosaccharide inhibitor GlcNAc (20 mM) was added during sWGA-lectin-affinity purification. Experiments were performed in duplicate (1 and 2). (B) Western blot analysis for Snail1 and O-GlcNAc from A549 cells in the presence/absence of OGA inhibitors PUGNAc or NAG-Thiazoline. α-tubulin was included as a loading control. (C) FLAG/HIS-tagged Snail1 was immunoprecipitated with Ni-Ti agarose from HEK293 cells and the precipitate analysed by western blot with anti-phospho-serine antibody and anti-FLAG antibody. Overexpression of OGT drastically reduces phospho-serine on Snail1. (D) FLAG-Snail1, HA-GSK-3β, and OGT were overexpressed in A549 cells as indicated. FLAG-Snail1 was immunoprecipitated and the precipitate immunoblotted with anti-HA antibody and anti-FLAG antibody, respectively. Arrow indicates HA-GSK-3β. (E) Half-life of Snail1 is increased by OGA inhibitors PUGNAc or NAG-Thiazoline. Cells were treated with cycloheximide for the indicated time and Snail1 level was analysed by western blotting and densitometry (n=3; data are represented as mean±s.d., ^*P<0.01, ^**P<0.02 by Student's t-test). (F) Western blot analysis for ubiquitin on Snail1 immunoprecipitated from HEK293 cells in the presence/absence of OGA inhibitor PUGNAc. The cells were treated with MG132 for proteasome inhibition. (G) Half-life of S112A mutant Snail1 in the presence/absence of OGA inhibitor PUGNAc. Cells were treated with cycloheximide for indicated time, and Snail1 level was analysed by western blotting. (H) Western blot analysis of wt and S112A mutant Snail1 in the presence/absence of OGA inhibitors PUGNAc or NAG-Thiazoline. α-tubulin was used as a loading control.