Journal: Journal of Biological Chemistry

Article Title: Mechanism of Ceftriaxone Induction of Excitatory Amino Acid Transporter-2 Expression and Glutamate Uptake in Primary Human Astrocytes

doi: 10.1074/jbc.m707697200

Figure Lengend Snippet: FIGURE 1. CEF induces EAAT2 expression in primary human fetal astro- cytes. PHFA cells were cultured in antibiotic-free medium for at least 7 days prior to all experiments reported in this study. Cells were treated with 10 M CEF for 2 days. A, cell lysates were prepared, and EAAT1 and EAAT2 protein expressions were analyzed. Actin was used as an internal control. B, total cellular RNA was extracted and subjected to Northern blotting for EAAT2 mRNA expression. C, nuclei were extracted, and the isolated nuclei were used to label preinitiated RNA transcription with [-32P]UTP in vitro, and the puri- fied RNA was hybridized to a dot blot carrying an equivalent amount of panel DNA probes. The transcript rate of glyceraldehyde-3-phosphate dehydro- genase (GAPDH) served as a control.

Article Snippet: Con- trol siRNA-A, EAAT1 siRNA, EAAT2 siRNA, and p65 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Cell Culture, Control, Northern Blot, Isolation, In Vitro, Dot Blot

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Ceftriaxone Induction of Excitatory Amino Acid Transporter-2 Expression and Glutamate Uptake in Primary Human Astrocytes

doi: 10.1074/jbc.m707697200

Figure Lengend Snippet: FIGURE2.CEFactivatestheEAAT2promoter.A,PHFAcellsweretransfected with the EAAT2Pro-954 together with a pSV--galactosidase plasmid as an internal control. One day after transfection, cells were treated with various concentrations of CEF for 2 days as indicated. Values are presented as -fold- normalized activity relative to that of the untreated cells taken as 1. B, PHFA cells were transfected with different EAAT2 promoter deletion-reporter con- structs with pSV--galactosidase as an internal control. One day after trans- fection, cells were treated with 10 M CEF for 2 days. Values are presented as -fold-normalized activity relative to that of the EAAT2Pro-954 in untreated cells taken as 1. Error bars indicate S.D.

Article Snippet: Con- trol siRNA-A, EAAT1 siRNA, EAAT2 siRNA, and p65 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Plasmid Preparation, Control, Transfection, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Ceftriaxone Induction of Excitatory Amino Acid Transporter-2 Expression and Glutamate Uptake in Primary Human Astrocytes

doi: 10.1074/jbc.m707697200

Figure Lengend Snippet: FIGURE 3. NF-B is crucial for CEF-induced EAAT2 expression. A, PHFA were infected with Ad.vec or Ad.IB- mt32. The infected cells (left) or uninfected PHFA (middle and right) were transfected with EAAT2Pro-954 together with pSV--galactosidase as an internal control. One day after transfection, cells were treated with 10 M CEF together with the indicated inhibitors (middle, 5 M quinalzoline or 20 M pyrrolidine dithiocarbamate; right, 5 M SN50M or SN50) for 2 days. Values are presented as –fold-normalized activity relative to that of the EAAT2Pro-954 in Ad.vec-infected (left), mock-treated (middle), or SN50M-treated (right) cells. B, PHFA were treated with 10 M CEF for 4 days, and nuclear extracts were prepared. Nuclear extracts were mixed with each radiolabeled oligonucleotide containing the NF-B binding site located at 583, 334, 272, or 251 of the EAAT2 promoter as follows: lanes 1, 3, 5, 7, and 9, untreated nuclear extracts; lanes 2, 4, 6, 8, and 10–13, CEF- treated nuclear extracts; lane 14, no extracts added. For competition assays, a 100-fold excess of corresponding unlabeled probe (lane 11) or unlabeled probe encompassing the mutated NF-B site (lane 12) was added. Supershift analysis was performed with an anti-p65 antibody (lane 13). The asterisk and arrow indicate, respec- tively, NF-B and a supershift band. C, PHFA were transfected with either the EAAT2Pro-954 or NF-Bmt-272 construct together with pSV--galactosidase as an internal control. One day after transfection, cells were treated with 10 M CEF for 2 days. Values are represented as –fold-normalized activity to that of the EAAT2Pro- 954 in untreated cells taken as 1. Error bars indicate S.D.

Article Snippet: Con- trol siRNA-A, EAAT1 siRNA, EAAT2 siRNA, and p65 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Infection, Transfection, Control, Activity Assay, Binding Assay, Construct

Journal: Journal of Biological Chemistry

Article Title: Mechanism of Ceftriaxone Induction of Excitatory Amino Acid Transporter-2 Expression and Glutamate Uptake in Primary Human Astrocytes

doi: 10.1074/jbc.m707697200

Figure Lengend Snippet: FIGURE 5. CEF induces glutamate uptake in PHFA. A, PHFA were trans- fected with control, EAAT1, or EAAT2 siRNA, and then the transfected cells were treated with 10 M CEF. Two days later, glutamate uptake levels were measured as described under “Experimental Procedures.” Accordingly, cell lysates from PHFA transfected with control, EAAT1, or EAAT2 siRNA were pre- pared and EAAT1 and EAAT2 expressions were analyzed. B, PHFA were infected with Ad.vec or Ad.IB-mt32, and then the uninfected or infected cells were treated with 10 M CEF. Two days later, glutamate uptake levels were measured. Error bars indicate S.D.

Article Snippet: Con- trol siRNA-A, EAAT1 siRNA, EAAT2 siRNA, and p65 siRNA were purchased from Santa Cruz Biotechnology.

Techniques: Control, Transfection, Infection