Figure 1.

Effects of neurons on NF-κB reporter activity and effects of dominant-negative NF-κB variants on induction of eGFP/GLT-1. A, Cortical astrocyte cultures from BAC GLT-1 eGFP transgenic mice were overlaid with a cell suspension from embryonic brain tissue obtained from wild-type animals (rats or mice) or a cell suspension containing astrocytes cultured from wild-type rat. In a subset of cultures, TTX (50 μm) or NMDA (1 mm) was added after 1 h. Cells were harvested after 10 d, and eGFP protein levels were analyzed. Data are representative of three independent experiments. B, Astrocyte cultures were infected with lentivirus engineered to express a NF-κB luciferase reporter and either dsRED (negative control) or dominant-negative constructs of IκBα (ΔN or SR). After 5 d, a fresh cell suspension containing neurons and astrocytes from wild-type E17 brain was overlaid on the top of infected astrocytes, and luciferase activity was measured after 10 d. Top, Summary of luciferase activity normalized to total protein content and expressed as a percentage of that measured in control astrocytes without neurons. Bottom, Western blot used to confirm expression of IκBα dominant-negative constructs in transduced astrocytes. Fifteen micrograms of cell lysate was loaded in each lane. Note that deletion of 36 aa in IκBα-ΔN construct results in faster migration of immunoreactive bands than in IκBα-SR. No other immunoreactive bands were observed in these blots. C, Top, Representative Western blot for eGFP protein. In all three experiments, eGFP was never detected in the absence of neurons and was observed in all experiments with neurons. Bottom, Graphic summary of data. D, GLT-1 protein levels were also analyzed in these same specimens. Top, A representative Western blot; bottom, summary of independent experiments. Data are the mean ± SEM of three independent experiments. *p < 0.05, ***p < 0.001 compared with corresponding astrocyte control; ^###p < 0.001 compared with astrocytes infected with vector control plus neurons.