Interaction of Nkx2.5 and <i>O-</i>GlcNAc transferase (OGT).

<p>(A) Immunoblotting analysis of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5 and OGT-flag-co-transfected HEK293 cells. (B) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in co-transfected cells and single transfected cells. <i>O</i>-GlcNAcylation levels were compared in no-transfected cells <i>vs</i> OGT-flag cells and myc-Nkx2.5 cells <i>vs</i> OGT-flag/myc-Nkx2.5 cells. Myc-Nkx2.5 protein levels were compared in myc-Nkx2.5 cells <i>vs</i> OGT-flag/myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05. (C) After immunoprecipitation with antibodies specific for Nkx2.5 and flag, immunoblotting with antibodies for myc, flag, and OGT was performed. (D) After immunoprecipitation with antibodies specific for OGT, immunoblotting with antibodies for myc, flag, and OGT was performed. The interaction between myc-Nkx2.5 and OGT-flag was observed.</p

Expression of Nkx2.5 in the heart tissue of diabetic mice (DM).

<p>(A) Immunoblotting analysis of Nkx2.5 and <i>O</i>-GlycNAcylated proteins in the heart tissue of DM intraperitoneally injected with streptozotocine (180 mg/kg body weight). (B) Graphs showed relative <i>O</i>-GlcNAc (left) and Nkx2.5 (right) levels normalized to the tubulin levels in DM and control. Nkx2.5 protein in diabetic heart of mice was significantly decreased. <i>O</i>-GlcNAcylation and Nkx2.5 protein levels were compared in DM <i>vs</i> control. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05 DM vs. control.</p

Reduction of myc-Nkx2.5 protein in cells treated with <i>O</i>-GlcNAcase inhibotirs.

<p>(A) Immunoblotting of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. <i>O</i>-GlcNAcylation levels were compared in untreated cells <i>vs</i> STZ-treated cells and untreated myc-Nkx2.5 cells <i>vs</i> STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of <i>O</i>-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-<i>N</i>-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the <i>O</i>-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. <i>O</i>-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05.</p

Modification of Nkx2.5 by <i>O</i>-GlcNAc.

<p>(A) The lysates of myc-Nkx2.5 and OGT-flag-co-transfected HEK293 cells were subjected to immunoprecipitation with anti-myc antibody and immunoblottings were performed with antibodies against <i>O</i>-GlcNAc (CTD110.6, RL-2) and myc. The modification of myc-Nkx2.5 with O-GlcNAc was detected. (B) Diabetic mice (DM) maintained elevated blood glucose level (>430 mg/dL) at 3 and 7 days after injection with streptozotocine (180 mg/kg body weight). The heart homogenates of control and diabetic mice were immunoprecipitated with anti-Nkx2.5 antibody, followed by immunoblottings with RL-2 antibodies against <i>O</i>-GlcNAc and anti-Nkx2.5 antibody. Nkx2.5 proteins of heart tissues were modified with <i>O</i>-GlcNAc and this modification increased in diabetic mice compared with control mice.</p