Fig. 2

TNF-α regulates GLS protein expression in mouse astrocytes. a Mouse astrocytes were treated with IL-1β (10 ng/ml) or TNF-α (50 ng/ml) for 72 h. Whole cell lysates were collected and protein levels of GLS, including two GLS allozymes, kidney type glutaminase (KGA, 65 kDa) and glutaminase C (GAC, 58 kDa), were analyzed by Western Blot. GFAP was a marker of astrocytes. Actin was used as a loading control. b Protein levels were normalized as a ratio to GFAP after densitometric quantification and presented as fold change relative to control astrocytes. c, d Intracellular concentration of glutamate was determined by a Glutamic Acid/Glutamate Oxidase Assay Kit, and the extracellular concentration of glutamate in cell-free supernatants was determined by HPLC. Results were expressed as the mean ± SD from three independent experiments. ** and *** denote p < 0.01 and p < 0.001 in comparison to control, respectively