Figure 2.

METH activates PKA and phosphorylates CREB at serine 133 (pCREB^Ser133). PKA enzyme activity in equivalent total cell lysates was quantified at select times post-METH treatment (500 μM, square and hatched lines) and represented as fold changes of PKA mUnits/mg total protein (A). PKA levels and activity were quantified in siCON- and siPKA-transfected astrocytes (clear and gray bars, respectively, as shown by fold changes in PKA/GAPDH mRNA (B), protein (C) and PKA activity (mUnits/mg total protein) (D). Immunoblotting for METH induction of pCREB^Ser133 to total CREB over time was assayed by western blot with detected bands at 43 kDa (E). To determine METH-induced pCREB^Ser133 via PKA, total cell lysates were collected at 30 min post-METH treatment in siCON- and siPKA-transfected astrocytes and immunoblotted for pCREB^Ser133 and total CREB. Bands are detected at 43 kDa for pCREB^Ser133 and total CREB (F). Cytoplasmic and nuclear protein extracts were collected from astrocytes treated with PKI (gray bars) +/– METH (hatched bars) and immunoblotted for pCREB^Ser133 (G). The same blot is represented in panel F & G from different sections; dividing lines represent cut sections. Representative western blots are shown in (E–G). Densitometry analyses were performed to quantify band intensities of phospho-proteins to total proteins on multiple immunoblots and represented as fold changes to control ± SEM, in respective panels [(E–G), n = 3]. (A–D) is a representative donor chosen from multiple individual biological astrocyte donors that were tested; each was analyzed in a minimum of triplicate determinations. Molecular weight markers are identified on each western blot (MW) (*p < 0.05, **p < 0.01, ***p < 0.001).