Journal: Circulation Research

Article Title: BMPER Is an Endothelial Cell Regulator and Controls Bone Morphogenetic Protein-4–Dependent Angiogenesis

doi: 10.1161/circresaha.108.178434

Figure Lengend Snippet: Figure 1. BMPER expression, localization, and regulation by FoxO3a in endothelial cells. A, Expression of BMPER in human vascular endothelial cells of different origin. Cells were lysed and subjected to Western blot analysis with the indicated antibodies. B and C, Localization of BMPER was characterized by immuno- cytochemistry in C166 mouse yolk sac endothelial cells. Corre- sponding serum was used as negative control. Nuclei were stained with DAPI. Scale bar100 m. D and E, Silencing of FoxO3a in HUVECs with 2 different siRNAs compared to scram- bled siRNA control resulted in enhanced BMPER expression shown by RT-PCR (D) and Western blot analysis (E). Seventy- two hours after transfection, mRNA expression was analyzed by using specific primers for FoxO3a, BMPER, and human RNA polymerase II. Western blot analysis was performed with the indicated antibodies. -Tubulin served as loading control. Rep- resentative Western blots are shown, along with densitometric analysis of the time course of BMPER expression. F, BMPER mRNA expression at 72 and 96 hours after transfection with FoxO3awt or the constitutively active mutant FoxO3aA3 com- pared to empty vector. BMPER mRNA was quantified by real- time (quantitative) PCR using specific primers for BMPER and hRP as internal control. BMPER mRNA expression was calcu- lated using the CT method. MeansSD. *P0.05 vs control.

Article Snippet: Total RNA was extracted from HUVEC using the Aurum RNA Mini Kit (Bio-Rad).

Techniques: Expressing, Western Blot, Immunocytochemistry, Negative Control, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Circulation Research

Article Title: BMPER Is an Endothelial Cell Regulator and Controls Bone Morphogenetic Protein-4–Dependent Angiogenesis

doi: 10.1161/circresaha.108.178434

Figure Lengend Snippet: Figure 2. Specific silencing of BMPER by siRNA in HUVECs. A, BMPER mRNA expression after 24 and 48 hours posttrans- fection with the siRNA BMPER I and II, respectively, compared to scrambled siRNA control. BMPER mRNA was quantified by real-time (quantitative) PCR using specific primers for BMPER and human RNA poly- merase II as internal control. Knockdown efficiency was calcu- lated using CT method. MeansSD; n4. *P0.001 vs con- trol. B, Representative semiquantitative RT-PCR analysis 24 hours posttransfection is shown. C, Western blot analysis was performed with the indicated antibodies 48 hours posttransfection.

Article Snippet: Total RNA was extracted from HUVEC using the Aurum RNA Mini Kit (Bio-Rad).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot