Figure 6.

HAND-relevant stimuli increase [Ca⁺²]_i, CaMKII activity and phosphorylation of CREB at serine 133 & 142. Primary human astrocytes were transfected with GCaMP6s. MOCK-transfected astrocytes were maintained as controls in parallel. Transfected cells were treated with IL-1β (20 ng/mL, A–C) or ionomycin (10 μM) as a positive control (images not shown). Fluorescence was visualized by confocal microscopy, and images were captured every 500 ms for 5 min. (A–C) depict astrocytes treated with IL-1 at selected time points. The histogram shows cumulative data, captured over 5 min (D). CaMKII activity was measured in astrocytes stimulated with IL-1β (20 ng/mL, hatched bars) for 30 min (E). Primary human astrocytes were treated with IL-1β and HIV-1 (blue bars), alone and in combination. Protein lysates were collected 30 min post-treatment and immunoblotted for total CREB, pCREB^Ser133, pCREB^Ser142, and GAPDH (F,G). Dividing lines on blots represented in (F,G) represent cut sections from the same blot. Representative western blots are shown in (F,G). Densitometry analyses were performed to quantify band intensities on multiple immunoblots and represented as fold changes to control ± SEM, in [(F,G), n = 3]. Statistical analyses were performed using GraphPad Prism V6.0 with one-way ANOVA and Tukey's post-test for multiple comparisons. P ≤ 0.05 were considered statistically significant, and data represent means ± SEM. This figure depicts representative donors chosen from multiple astrocyte donors that were tested; each was analyzed in a minimum of triplicate determinations. Data are shown as cumulative fold changes. n represents individual biological replicates. Molecular weight markers are identified on each western blot (MW) (*p < 0.05, **p < 0.01, ***p < 0.001).