Fig. 1.Concentration-dependent increase in protein O-GlcNAc levels in
neurons from rTg4510 mice treated with Thiamet G. Cortical neurons isolated from
rTg4510 mouse embryos were incubated at days in vitro
(DIV) 7 with Thiamet G at concentrations as indicated. After 6
hours, neurons were lysed and analyzed by western blot for global protein
O-GlcNAcylation (O-GlcNAc), human tau (E1), total tau (Tau5), and phosphorylated
tau at S199/202 (pS199/202) and S396 (pS396) epitopes. For normalization, the
O-GlcNAC blot was re-probed with GAPDH. The E1 blot was re-probed with pS396 and
GAPDH, and the pS199/202 blot was re-probed with Tau5 and GAPDH. Phosphorylated
and non-phosphorylated human 0N4R transgene tau was displayed at 55 kDa with the
E1 antibody. Murine and human 0N4R transgene tau species are displayed at 45 and
55 kDa, respectively, with the Tau5 antibody. (A) Representative western blots
from n = 3 experiments. (B) Quantification of western blots is shown as graphs in
absolute units O-GlcNAc/GAPDH and in % tau/GAPDH with buffer control (0) set to
100%. Individual samples are presented as filled shapes. Thiamet G EC
based on densitometric data from total O-GlcNAcylation (O-GlcNAc) western blot
presented in A was calculated to 33 nM. GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.