Figure 4.

METH activates CaMKII and phosphorylates CREB at serine 142 (pCREB^Ser142). CaMKII enzyme activity was quantified at 5, 15, and 30 min post-METH treatment (500 μM, squares and hatched bars) in equal amounts of cell lysates and represented as fold changes of CaMKII mUnits/mg total protein (A). CaMKII levels and activity were quantified in siCON- and siCaMKII-transfected astrocytes (clear and gray bars, respectively), and represented as CaMKII/GAPDH fold changes and fold changes of CaMKII mUnits/mg total protein (B,C). To evaluate METH-induced phosphorylation of CaMKII, immunoblotting for pCaMKII total CaMKII and GAPDH was detected at 54, 50, and 37 kDa, respectively in astrocytes treated with METH (500 μM) at different time points (D). Immunoblotting for pCREB^Ser142 to total CREB over time was assayed by western blot with detected bands at 43 kDa (E). To determine METH-induced pCREB^Ser142 via CaMKII, total cell lysates were collected at 30 min post-METH treatment in siCON- and siCaMKII-transfected astrocytes and immunoblotted for pCREB^Ser142 and total CREB (F). The same blot is represented in (F) from different sections, dividing line represents a cut section. Representative donors are shown in all panels from multiple astrocyte donors that were tested; each was analyzed in a minimum of triplicate determinations. Densitometry analyses of ratio for phospho-proteins to total proteins was performed to quantify band intensities on multiple immunoblots and represented as fold changes to control ± SEM [(D–F), n = 3]. Molecular weight markers are identified on each western blot (MW) (*p < 0.05, **p < 0.01, ***p < 0.001).