Journal:

Article Title: Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers

doi: 10.1523/JNEUROSCI.0311-10.2010

Figure Lengend Snippet: (A) Kv1.3 gene expression was detected using real-time PCR in NPC cultures treated with GrB (4 nM) for 1-3 hours. GADPH was used as an internal control. Dose-dependent increase in Kv1.3 mRNA expression was noted. Data represent mean ± SEM from four independent experiments. (B) NPC cultures were treated with GrB (4nM) for 24 hours and immunostained with antibodies to Kv1.3 and nestin (Red: nestin, Green: Kv1.3). (C) NPC cultures were treated with GrB (1-4 nM) for 24 hours and the cell lysates were collected for detecting Kv1.3 production using western-blot analysis. This shows a dose-dependent increase in Kv1.3 detection.

Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal:

Article Title: Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers

doi: 10.1523/JNEUROSCI.0311-10.2010

Figure Lengend Snippet: To monitor NPC neurogenesis NPC cultures were cultured in differentiating media for 4-7 days. Cells were immunostained with anti-beta-III tubulin antibody (red) and anti-GFAP antiserum (green). Beta-III tubulin positive neurons were counted in each well and the cell numbers were expressed as percentage of total cells. Cells in 5 pre-assigned fields (approx 200 cells/field) were counted on each cover slip and three cover slips were counted in every group. Data represents mean ± SEM from three experiments. (A) Kv1.3 selective blocker MgTX (10 nM) prevented the effects of GrB (4 nM) on NPC neurogenesis. (B) MgTX also attenuated the effect of supernatants from activated T cells (Ac-T) on NPC neurogenesis. (C) Transfection of Kv1.3 siRNA (25 nM final concentration) but not nonspecific control (NSi) into NPC blocked the effects of GrB (4 nM) on NPC neurogenesis. (D) Representative photomicrographs immunostaining for beta-III tubulin.

Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

Techniques: Cell Culture, Transfection, Concentration Assay, Immunostaining

Journal:

Article Title: Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers

doi: 10.1523/JNEUROSCI.0311-10.2010

Figure Lengend Snippet: Eight weeks old female rats were stereotaxically injected in the dentate gyrus (DG) with GrB (1 ug), MgTX (10 ng) +GrB (1 ug) or vehicle control (PBS). After seven days, the rats received BrdU (100 mg/kg, i.p.) two hours before being euthanized. Serial sections of the hippocampus and DG were analyzed by quantitative immunohistochemistry. Sections were immunostained with anti-BrdU (red), and anti-NeuN (green) or anti-Kv1.3 (green). DAPI (blue) was used for nuclear staining. BrdU positive cells in the subgranular zone (SGZ) of every sixth section were counted in a blinded fashion, and normalized to the volume of each granule cell layer (GCL). (A) Representative immunostained sections are shown from each of the treated groups. (B) Quantitative analysis shows decreased numbers of BrdU staining cells with GrB and restoration with MgTX (C) Kv1.3 (green) fluorescence immunohistochemistry shows GrB increases Kv1.3 expression in the dentate gyrus compared to vehicle control. The cellular localization of Kv1.3 (green) was characterized by co-localization studies with BrdU (red).

Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

Techniques: Injection, Immunohistochemistry, Staining, BrdU Staining, Fluorescence, Expressing

Journal:

Article Title: Activated T cells Inhibit Neurogenesis by Releasing Granzyme B: Rescue by Kv1.3 blockers

doi: 10.1523/JNEUROSCI.0311-10.2010

Figure Lengend Snippet: NPC cultures were pretreated with PTX for 1 h prior to GrB treatment. The cells were lysed 3 h after GrB treatment. Kv1.3 mRNA was detected using real-time PCR. The results were expressed as folds compared to control, Data were from three independent experiments.

Article Snippet: Kv1.3 expression was also monitored in NPC cells by immunocytochemistry and western blot using anti-Kv1.3 polyclonal serum (1: 100, Alomone Labs, Jerusalem, Israel).

Techniques: Real-time Polymerase Chain Reaction