Table 1.
Primary antibodies employed in this study.
Figure 1.
Experimental design and effects of icv injection of thiamet-G on brain protein O-GlcNAcylation.
(A) Diagram showing the experimental design of icv injection. Thiamet-G (175 µg per mouse) was injected into the lateral ventricles of the brain, and then the tau Tg mice were sacrificed 4.5 h, 9 h, or 24 h after injection. (B) The brain homogenates were analyzed by Western blots developed with monoclonal antibody RL2 against O-GlcNAcylated proteins and, as a loading control, anti-GAPDH. (C) All of the RL2-positive bands in the blots were quantified densitometrically, and the intensities of the total O-GlcNAcylated proteins after being normalized by the intensity of GAPDH are shown (mean ± SEM; n = 5; *, p<0.05; **, p<0.01; ***, p<0.001 vs. saline controls).
Figure 2.
Levels of phosphorylation of tau at different sites in tau Tg mouse brains after icv injection of thiamet-G.
(A) Western blots of the brain homogenates developed with an antibody to total tau (92e) and a battery of phosphorylation-dependent and site-specific antibodies to determine tau phosphorylation at individual phosphorylation sites, as indicated at the left side of the blots. (B–D) Levels of tau phosphorylation at individual sites 4.5 h (B), 9 h (C), or 24 h (D) after injection were determined by densitometrical quantitation of the blots, as shown in A. Data (mean ± SEM; n = 5) are presented as percentage of saline control injection. The total tau level of each sample was used for normalization for calculation. *p<0.05 vs. saline injection.
Figure 3.
Analyses of major tau kinases in tau Tg mouse brains after thiamet-G treatments.
(A) Western blots of the brain homogenates developed with antibodies indicated at the left side of the blots. GAPDH blot was included as a loading control. (B) The blots were quantified densitometrically, and the immunoreactivities (mean ± SEM; n = 5) of the indicated phosphorylated kinases over that of the total kinase counterparts are shown to represent the activation statuses of these kinases. Note that the GSK-3β(Y216) antibody also recognized GSK-3α (upper band in A). Only the GSK-3β band (lower band) was used for quantification. In case of CDK and p35, GAPDH was used for normalization. *p<0.05 vs. saline injection.
Figure 4.
Effects of thiamet-G treatment on protein O-GlcNAcylation and tau phosphorylation in AHP cells.
(A) Proliferating AHP cells were treated with 20 nM thiamet-G for various periods of time, and the protein O-GlcNAcylation of the cell lysates was determined by Western blots developed with RL-2 against O-GlcNAc. (B) The blots were quantified densitometrically, and the O-GlcNAc levels are presented as the percentage of control cells at each time point. (C) Western blots developed with 92e for detecting total tau level and with several phosphorylation-dependent and site-specific tau antibodies for detecting phosphorylation of tau at individual sites. (D) The tau blots were quantified densitometrically, and the tau level and site specific phosphorylation level are presented as the percentage of control cells at each time point. The data shown are mean ± SEM of triplicates of one of three separate experiments with similar results.