FIGURE 4.

Activation of sodium-dependent glutamate transporters leads to a transient phosphorylation event at CaMKIIβ’s S³⁷¹ site. A: Bar graph depicting a quantitative analysis of the oligodendrocyte network area (Dennis et al. 2008). Cells were pre-treated with the pharmacological CaMKII inhibitor KN-93 or its inactive derivative KN-92 and then incubated in the absence or presence (+Glu) of 100 μM L-glutamate. The mean values for control cells (pre-treated with KN-92 and incubated in the absence of L-glutamate) were set to 100% and experimental values were calculated accordingly. Data represent means ± SEM (***p≤0.001 compared to control, ANOVA). The inset (upper right) depicts representative images of differentiating oligodendrocytes treated with KN-92 plus L-glutamate (left) or KN-93 plus L-glutamate (right) and immunostained using O4 hybridoma supernatants. Scale bars: 5 μm. B, inset in H: Representative Western blots depicting CaMKII phosphorylation (pCaMKIIβ S³⁷¹, pCaMKII T^286/7) or total CaMKIIβ protein levels. GAPDH protein levels are shown representatively for the Western blot for which anti-pCaMKII T^286/7 (B) or anti-pCaMKIIβ S³⁷¹ (inset in H) antibodies were used. C-H: Bar graphs depicting the levels of pCaMKIIβ S³⁷¹ (C,F- H), total CaMKIIβ (D) or pCaMKII T^286/7 (E) at different time-points after addition of L-glutamate (Glu) (C-F), at the time-point of 60 min after addition of L-glutamate and prior pre-treatment with or without TBOA (G) or after transfection with siRNA pools as indicated and L-glutamate treatment for 60 min (H). All CaMKII protein levels were normalized to GAPDH protein levels. The mean normalized values for control (non-treated) cells were set to 100% (horizontal line) and experimental values were calculated accordingly. Data represent means ± SEM of 3 independent experiments (***p≤0.001, *p≤0.05 compared to control, ANOVA).