Fig. 5

OGA inhibition by thiamet G (TG) increases MTOR activation. (a) Primary cortical neurons were cultured from E18 rat embryos. At DIV7, neurons were exposed to TG (0.25 μM) for 24 h. Western blot analyses were performed with anti-p-MTOR (at Ser2448) and anti-MTOR antibodies. Bar graphs represent respective quantifications. (b-d) DIV7 primary rat cortical neurons were exposed to rapamycin (1 μM) and/or TG (0.25 μM) for 7 d. Western blot analyses were performed. β-actin was used as a loading control. (b) Inhibition of OGA by TG (7 d) increased p-MTOR/total MTOR and rapamycin decreased it. Western blot analyses of p-MTOR (at Ser2448), and MTOR were performed. (c) TG (7 d) increased total O-GlcNAcylated proteins even in the presence of rapamycin. Western blot analyses of O-GlcNAcylated proteins were performed. (d) Thiamet G and rapamycin both decreases O-GlcNAc transferase (OGT), as shown by the western blot analyses. Quantification of western blot band intensities was performed using NIH image J. For all panels, Data = mean ± SEM (n = 3), *p < 0.05 compared to no TG, #p < 0.05 compared to –rapamycin. Results were analyzed by ANOVA followed by Bonferroni’s Multiple Comparison post-hoc test