Journal: The Journal of Biological Chemistry

Article Title: GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling *

doi: 10.1074/jbc.M114.571000

Figure Lengend Snippet: Primers for RT-PCR/qPCR assay F is forward primer; R is reverse primer; M is Mus musculus ; and D is Danio rerio .

Article Snippet: The control siRNA for STAT5 and GATA2 is a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA afforded by the GenePharma Co. For the cell growth curve assay, cells were seeded at 1 × 10 4 cells/well and grown in 12-well plates continuously for 1–7 days in triplicate.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling *

doi: 10.1074/jbc.M114.571000

Figure Lengend Snippet: Primers for PCR in chromatin immunoprecipitation assays

Article Snippet: The control siRNA for STAT5 and GATA2 is a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA afforded by the GenePharma Co. For the cell growth curve assay, cells were seeded at 1 × 10 4 cells/well and grown in 12-well plates continuously for 1–7 days in triplicate.

Techniques: Chromatin Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling *

doi: 10.1074/jbc.M114.571000

Figure Lengend Snippet: GPR126 stimulates VEGFR2 transcription via STAT5 and GATA2. A, effects of GPR126 on the expression of transcription factors involved in angiogenesis. Protein levels were analyzed by immunoblot using specific antibodies in HMEC-1 cells (Con) and in cells infected with vector control (NC) or GPR126 shRNA (Sh1 and Sh2). B and C, regulation of VEGFR2 expression by GATA2. VEGFR2 protein level was analyzed by immunoblot in HMEC-1 cells (Con), cells transfected with control siRNA (Scramble), or GATA2 siRNA (GATA2 si1 and GATA2 si2) (B). Chromatin of HMEC-1 cells was immunoprecipitated with anti-GATA2 antibody or IgG control. The extracted DNA was used for PCR amplifications with VEGFR2 promoter-specific primers (C). D–F, regulation of VEGFR2 expression by STAT5. VEGFR2 protein level was analyzed by immunoblot in HMEC-1 cells (Con), cells transfected with control shRNA (NC), or STAT5 shRNA vector (STAT5Sh) (D), or control siRNA (Scramble), and STAT5 siRNA (ST5 si1 and ST5 si2) (E), respectively. F, forced expression of STAT5 increased VEGFR2 expression in HMEC-1 cells. Zs, control vector; ZsSTAT5, STAT5 overexpression vector. G, chromatin of HMEC-1 cells was immunoprecipitated with anti-STAT5 antibody or IgG control. The extracted DNA was used for PCR amplifications with VEGFR2 promoter-specific primers. STAT5 strongly bound to the predicted site (−3285 to −3277, TTCTGTGAA) in the VEGFR2 promoter. The assays were conducted at least three times. H, wild type (WT-Luc) or STAT5-binding site mutant (Mut-Luc) VEGFR2 promoter luciferase reporter was transfected with increasing doses of STAT5 expression plasmid, and the luciferase activity was determined. I, in vitro translated STAT5 protein was incubated with hot STAT5-binding element derived from VEGFR2 for EMSAs. Cold WT (Cold) or mutant STAT5-binding site (M-probe) probes were subjected for competition. J–L, GPR126 regulated STAT5 and GATA2 expression through cAMP-activated PKA-CREB pathway. Phospho-CREB (Ser-133) was activated in HMEC-1 cell with forskolin (20 μm) stimulation and inhibited with H-89 (10 μm) treatment (J). K, forskolin increased the STAT5 and GATA2 protein levels in a dose-dependent manner in HMEC-1 cells. L, ChIP assays of the CRE site in the STAT5 and GATA2 promoter. Top panel showed the predicted conserved CRE site or half-CRE site in the STAT5 promoter. Middle panel showed that of the GATA2 promoter. After immunoprecipitation of the cross-linked complexes, DNA was recovered by phenol/chloroform extraction. Then the DNA were amplified by PCR using indicated primers. The PCR bands in bottom panel showed the DNA fragment precipitated by anti-CREB antibody in the promoter region of STAT5 (bottom left) and GATA2 (bottom right), respectively.

Article Snippet: The control siRNA for STAT5 and GATA2 is a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA afforded by the GenePharma Co. For the cell growth curve assay, cells were seeded at 1 × 10 4 cells/well and grown in 12-well plates continuously for 1–7 days in triplicate.

Techniques: Expressing, Western Blot, Infection, Plasmid Preparation, Control, shRNA, Transfection, Immunoprecipitation, Over Expression, Binding Assay, Mutagenesis, Luciferase, Activity Assay, In Vitro, Incubation, Derivative Assay, Extraction, Amplification

Journal: The Journal of Biological Chemistry

Article Title: GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling *

doi: 10.1074/jbc.M114.571000

Figure Lengend Snippet: STAT5 and GATA2 restored the angiogenic activity of ECs attenuated by GPR126 knockdown. A, forced expression of STAT5 and/or GATA2 in GPR126 knockdown HMEC-1 cells restored two-dimensional tube formation on Matrigels. pll3.7, control vector of shRNA; Zs, control vector for overexpression; GPR126-Sh, GPR126 shRNA; ST5, STAT5 overexpression; GA2, GATA2 overexpression. B, statistical summary of relative tube length of each group in A. C, restoration of angiogenic activity of endothelial cells in zebrafish embryos. Bright field images showed the morphology of 30 hpf Tg(fli1:EGFP)y1 zebrafish embryos in each injection treatment. Confocal fluorescence images showed ISVs sprouting of Tg(fli1:EGFP)y1 embryos of each treatment. D, statistical summary of percentage of embryos with ISV defect in each group in C. The numbers above the column represent the number of embryos with ISV defect/the number of embryos analyzed totally.

Article Snippet: The control siRNA for STAT5 and GATA2 is a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA afforded by the GenePharma Co. For the cell growth curve assay, cells were seeded at 1 × 10 4 cells/well and grown in 12-well plates continuously for 1–7 days in triplicate.

Techniques: Activity Assay, Knockdown, Expressing, Control, Plasmid Preparation, shRNA, Over Expression, Injection, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling *

doi: 10.1074/jbc.M114.571000

Figure Lengend Snippet: Diagram of putative mechanisms of GPR126 function in endothelial cells. GPR126 stimulates STAT5 and GATA2 transcriptional activities through cAMP-activated PKA-CREB pathway. The activated STAT5 and GATA2 independently bind to the VEGFR2 promoter and activate its expression. Up-regulation of VEGFR2 protein levels amplifies downstream angiogenic cascades, such as activation of FAK and ERK. Thus, GPR126 promotes VEGF signaling and angiogenesis by modulating VEGFR2 expression through STAT5 and GATA2 in endothelial cells.

Article Snippet: The control siRNA for STAT5 and GATA2 is a scrambled sequence that will not lead to the specific degradation of any known cellular mRNA afforded by the GenePharma Co. For the cell growth curve assay, cells were seeded at 1 × 10 4 cells/well and grown in 12-well plates continuously for 1–7 days in triplicate.

Techniques: Expressing, Activation Assay