Journal: The Journal of Neuroscience
Article Title: Sox21 Promotes Hippocampal Adult Neurogenesis via the Transcriptional Repression of the Hes5 Gene
Figure Lengend Snippet: Repression of Hes5 gene promoter activity by Sox21 but not Sox2. AHP cells were transfected with Hes5 promoter–luciferase reporter constructs (1 μg per well) with or without expression vectors for NICD , Sox21 , and/or Sox2 . The histogram shows the ratio of firefly luciferase to that in cells transfected with the reporter construct and the NICD -expressing vector. The transfection efficiency was normalized to Renilla luciferase activity. A , B , The Hes5 DRE and proximal promoter (−2767 to +73 bp from TSS) and the proximal promoter alone (−687 to +73 bp) were used as reporter constructs in A and B , respectively. The open columns in A show the relative luciferase activities using the intact reporter construct, and the closed columns reflect the construct mutagenized at the Sox-binding sequence of the DRE (ACAAAGG to AagcttG). NICD -expression vector, 0.1 μg per well; Sox21 -expression vector, 0.0001, 0.001, and 0.01 μg per well. C , The Hes5 DRE (−2676 to −2244 bp) combined with the β-globin minimal promoter was used. The open columns indicate the intact reporter construct, and the closed columns indicate the mutagenized construct as shown in A . NICD, 1 μg per well; Sox21, 0.0001, 0.001, and 0.01 μg per well. D , E , The Hes5 DRE and proximal promoter were used. D , NICD, 0.1 μg per well, Sox2, 0.0001, 0.001, and 0.01 μg per well. E , NICD, 0.1 μg per well; Sox21, 0.01 μg per well; Sox2, 0.0001, 0.001, and 0.01 μg per well. Data represent the mean ± SE. Statistical significance versus the intact promoter was assessed by two-sided t test ( n > 4). ** p
Article Snippet: The following primary antibodies were used in this study: anti-βIII-tubulin (rabbit IgG; Covance, PRB-435P; 1:500), anti-glial fibrillary acidic protein (GFAP; rabbit IgG; DAKO, Z0334; 1:500), anti-GFAP (mouse IgG; Sigma-Aldrich, G3893; 1:200), anti-fatty acid binding protein 7 (FABP7; rabbit IgG; Millipore, AB9558; 1:250), anti-Sox21 (goat IgG; R & D Systems, AF3538; 1:100), anti-Sox2 (rabbit IgG; Millipore, AB5603; 1:200), anti-Sox1/(2)/3 (rabbit IgG; gift from Dr. H. Kondoh, Osaka University, Osaka, Japan; 1:5000) , anti-Nestin (mouse IgG; BD Biosciences/PharMingen, 556309; 1:200), anti-doublecortin (DCX; goat IgG; Santa Cruz Biotechnology, sc-8066; 1:100), anti- DCX (rabbit IgG; Abcam, ab18723; 1:100), anti-polysialylated neuronal cell adhesion molecule (PSA-NCAM; mouse IgMκ; gift from Dr. T. Seki, Tokyo Medical University, Tokyo, Japan; 1:200), anti-NeuN (mouse IgG; Millipore, MAB377; 1:100), anti-S100β (mouse IgG; Sigma-Aldrich, S2532; 1:200), anti-glutathione S -transferase π (mouse IgG; BD Biosciences/PharMingen, 610719; 1:200), anti-Brn2 (Santa Cruz Biotechnology, sc-6029; 1:100), anti-Tbr1 (rabbit IgG; gift from Dr. R. F. Hevner, University of Washington, Seattle, WA; 1:10,000), anti-Ascl1 (mouse IgG; BD Biosciences/PharMingen, 556604; 1:100), anti-Tbr2 (rabbit IgG; Abcam, ab23345; 1:200), anti-Ki67 (rabbit IgG; Abcam, ab15580; 1:3000), anti-GFP (rabbit IgG, Medical and Biological Laboratories, 598, 1:500; goat IgG, Rockland, 600-101-215, 1:500), anti-BrdU (rat IgG; Abcam, ab6326; 1:100), anti-lacZ (rabbit IgG, Cappel, 55976, 1:500; goat IgG, Cappel, 56028, 1:500), and normal IgG (rabbit IgG, Millipore, DAM1421465; goat IgG, Santa Cruz Biotechnology, sc-2028).
Techniques: Activity Assay, Transfection, Luciferase, Construct, Expressing, Plasmid Preparation, Binding Assay, Sequencing