Journal: PLoS ONE

Article Title: Dietary Restriction Affects Neuronal Response Property and GABA Synthesis in the Primary Visual Cortex

doi: 10.1371/journal.pone.0149004

Figure Lengend Snippet: The top panel shows a typical sample of a Western blot result from each DR cat (DR1-4) and control cat (NC1-4) using GAD67 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies. The bottom panel shows the average optical density (OD) of GAD67 bands normalized against the corresponding GAPDH in both groups of cats. The average normalized optical density of GAD67 in DR cats was significantly higher than that in the control (** p<0.01).

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-Tween 20 for one hour and incubated overnight at 4°C in TBS-Tween 20 containing a rabbit polyclonal antibody GAD67 (dilution 1:200; Boster Bio-engineering Limited Company) or a rabbit polyclonal antibody GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (dilution 1:5,000; Sangon Biotechnology).

Techniques: Western Blot, Control

Journal: Cells

Article Title: TWIK-1 BAC-GFP Transgenic Mice, an Animal Model for TWIK-1 Expression

doi: 10.3390/cells10102751

Figure Lengend Snippet: Cellular identification of GFP-expressing cells of the DG, LEC, and Cb in P56 of TWIK-1 BAC-GFP Tg mice. ( A ) Overview of GFP expression in DG, LEC, and CB of TWIK-1 BAC-GFP Tg mice. Scale bar, 200 μm. ( B ) Representative co-immunofluorescence images with GFP, NeuN, and GAD67 antibodies. Scale bar, 200 μm. ( C ) Quantification bar graph of the cell type of GFP-positive cells in each brain area from B. Quantification was analyzed by the percentage of each cell type from all GFP-positive cells. Raw data are listed in . Data are presented as the Mean ± SEM.

Article Snippet: The following antibodies were used: chicken anti-GFP (Abcam, Cat#; ab136970, 1:300); rabbit anti-TWIK-1 (Alomone labs, Cat#; APC-10, 1:200); mouse anti-NeuN (Abcam, Cat#; ab104224, 1:100); rabbit anti-GAD67 (GeneTex, Cat#; GTX113190, 1:300); guinea pig anti-doublecortin (Millipore, Cat#; AB2253, 1:100); rabbit anti-calbindin (Swant, Cat#; CB38, 1:2000); rat anti-Ki67 (Invitrogen, Cat#; 14-5698-85, 1:200); rat anti-GFAP (Invitrogen, Cat#; 13-0300, 1:500); rabbit anti-NG2 (Millipore, Cat#; AB5320, 1:300); and Alexa Fluor 488-, 594-, and 647-conjugated secondary antibodies (Jackson ImmunoResearch, 1:300).

Techniques: Expressing, Immunofluorescence

Journal: PLoS ONE

Article Title: Role of the Proteasome in Excitotoxicity-Induced Cleavage of Glutamic Acid Decarboxylase in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0010139

Figure Lengend Snippet: Neurons were stimulated with 125 µM glutamate, for 20 min, and further incubated in culture conditioned medium for the indicated period of time. Full length GAD 65/67 protein levels were determined by Western Blot with an antibody that recognizes both isoforms. Control protein levels of GAD65/67 were set to 100%. Actin was used as loading control (A). Panel A shows a representative experiment and mean±SEM of 9 independent experiments. The cleavage of GAD67 was also analysed with an antibody directed against amino acids 70–130 of this isoform (B). In this case the results obtained under control conditions were compared with the immunoreactivity in extracts prepared 14 h after the toxic insult. The amino acid sequence of GAD65 (lower sequence) and 67 (top sequence) are aligned in panel C, which also show the binding sites for the antibodies used in this study. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni's multiple comparison test. ** p <0.01; *** p <0.001.

Article Snippet: The following primary antibodies were used: anti-GAD65/67 (1∶5000, Sigma), anti-GAD67 (1∶250; BD Biosciences, Erembodegem, Belgium), antibody against calpain-mediated fragment of spectrin/fodrin nSBDP N SBDPs [1∶300 , ] and anti-β-Actin (1∶5000, Sigma).

Techniques: Incubation, Western Blot, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: Role of the Proteasome in Excitotoxicity-Induced Cleavage of Glutamic Acid Decarboxylase in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0010139

Figure Lengend Snippet: Cultured hippocampal neurons were pre-incubated or not with 50 µM of UBEI-41 (E1 inhibitor), for 1h, or with the proteasome inhibitors MG132 (1 µM), lactacystin (10 µM) or YU102 (10 µM), for 30 min, before excitotoxic stimulation with glutamate (125 µM), for 20 min. The cells were further incubated in culture conditioned medium (with or without chemical inhibitors) for 5h, and the GAD65/67 immunoreactivity was assessed by western blot (A and B). Incubation with the calpain inhibitor ALLN (10 µM) was performed under the same conditions. The average results in (A) represent the changes in GAD65 or GAD67 immunoreactivity. In panel (B) GAD cleavage was calculated as a percentage of the total enzyme content (GAD65/67). When calpain activity was evaluated through formation of N-terminal spectrin breakdown products ( N SBDPs) the cells were incubated for 30 min after the toxic insult (C). The effect of MG132 and UBEI-41 on calpain activation is expressed as a percentage of the activity measured in the absence of the protease inhibitors. Results are means±SEM of 3–4 different experiments, performed in independent preparations. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni's Multiple Comparison Test (A and B) or the Student's t test (C). *** p <0.001.

Article Snippet: The following primary antibodies were used: anti-GAD65/67 (1∶5000, Sigma), anti-GAD67 (1∶250; BD Biosciences, Erembodegem, Belgium), antibody against calpain-mediated fragment of spectrin/fodrin nSBDP N SBDPs [1∶300 , ] and anti-β-Actin (1∶5000, Sigma).

Techniques: Cell Culture, Incubation, Western Blot, Activity Assay, Activation Assay