Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 2 Effect of DCN on expression and activation of AKT, EGFR, HIF1a, Stat3, and Sp1 in DCN transfected cerebral endothelial cells. (a) Quantitative real-time PCR (top of the gel) and western blot (gel) for AKT are analyzed from the samples (shown in corresponding lanes at the bottom) of mock transfected (control) and three DCN transfected clones (D4, D9, and D10) in five independent experiments. The loading of the samples is normalized with b-actin. (b) Western blot (gel) for phosphorylated AKT (P-AKT) is analyzed from the samples (shown in corresponding lanes at the bottom) of mock transfected (control) and three DCN transfected clones (D4, D9, and D10). Each column represents the mean percentage of phosphorylated AKT out of total AKT of five samples. Total AKT and P-AKT were quantified by histogram values of individual bands subtracting from their back- ground without a band. (c) Effect of AG1478, LY294002, and PTEN transfection are analyzed in western blot from the protein samples of control and DCN transfected MCE clone D10 (MCE-D10) (shown in the corresponding lanes at the top).

Article Snippet: The HIF1a and Stat3 probes were retarded with the nuclear extracts from DCN synthesizing MCE cells and the nuclear extracts pre-incubated with 100% excess of Sp1 probes.

Techniques: Expressing, Activation Assay, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Control, Clone Assay

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 3 AG1478 and LY294002 treatments inhibit DCN-mediated binding of HIF1a, Stat3, and Sp1 on the VEGF promoter. (a–c) Binding of transcription factors to their specific motifs on the VEGF promoter is analyzed by gel shift assay for the samples from mock transfected MCE cells (MCE-Control), DCN transfected MCE clone D10 (MCE- D10), and MCE-D10 subjected to the different treatments shown in the corresponding lanes. Arrows indicate the retarded probes and un- bound free probes. The transcription factor binding motifs in VEGF promoter are used as their specific oligonucleotide probes in the VEGF promoter, shown at the bottom of the gel. (d) The transcription factors binding specificity is confirmed by super shift analysis. Arrows indicate the retarded probes and super shifted with their specific antibodies (Ab). The samples from DCN transfected MCE cells are pre-incubated with their probes (shown at the bottom of the gel) their specific antibodies (shown in the corresponding lane at the top of the gel).

Article Snippet: The HIF1a and Stat3 probes were retarded with the nuclear extracts from DCN synthesizing MCE cells and the nuclear extracts pre-incubated with 100% excess of Sp1 probes.

Techniques: Binding Assay, Gel Shift, Transfection, Control, Incubation

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 4 Quantitative sequential chromatin immunoprecipitation (SChIP) analysis of binding of Sp1, HIF1a, and Stat3 to the VEGF promoter in vivo in DCN synthesizing MCE cells followed by AG1478, LY294002, and PTEN treatment. (a) The locations of the two pairs of primer sets in human VEGF promoter are used to detect the ChIP amplified DNA fragment (190 bp), indicated as filled bar containing Sp1motif (open bar) and DNA fragment (290 bp) indicated as filled bar containing HIF1a (HRE) and Stat3 motifs (open bars). (b) SChIP was performed for the chromatin samples of the corresponding lanes shown in the bottom of the gel by using Sp1, HIF1a, and Stat3 anti- bodies (shown in bottom). Bar graph indicates the fold enrichment that is the relative abundance of DNA fragments at the indicated regions over a control region as quantified by real-time PCR. Migration of PCR products and protein in western blot from SChIP analysis are marked in left. Bar graph at top indicates fold enrichment of SChIP analysis of corresponding samples.

Article Snippet: The HIF1a and Stat3 probes were retarded with the nuclear extracts from DCN synthesizing MCE cells and the nuclear extracts pre-incubated with 100% excess of Sp1 probes.

Techniques: Chromatin Immunoprecipitation, Binding Assay, In Vivo, Control, Real-time Polymerase Chain Reaction, Migration, Western Blot

Journal: Journal of neurochemistry

Article Title: Ectopic decorin expression up-regulates VEGF expression in mouse cerebral endothelial cells via activation of the transcription factors Sp1, HIF1alpha, and Stat3.

doi: 10.1111/j.1471-4159.2007.05134.x

Figure Lengend Snippet: Fig. 9 Schematic presentation of pathways of VEGF promoter regu- lation by EGFR/DCN. Activation of Sp1, HIF1a, and Stat3 via DCN/ EGFR/PI3K/AKT and DCN/EGFR/ERK1/2 pathways and eventually activation of VEGF promoter by binding to their consensus motifs into the VEGF promoter are indicated by arrows.

Article Snippet: The HIF1a and Stat3 probes were retarded with the nuclear extracts from DCN synthesizing MCE cells and the nuclear extracts pre-incubated with 100% excess of Sp1 probes.

Techniques: Activation Assay, Binding Assay