dr3 tnfrsf25 goat anti mouse  (R&D Systems)

Journal: International journal of oncology

Article Title: Epigenetic modulators hydralazine and sodium valproate act synergistically in VEGI-mediated anti-angiogenesis and VEGF interference in human osteosarcoma and vascular endothelial cells.

doi: 10.3892/ijo.2019.4811

Figure Lengend Snippet: Figure 2. Effects of Hy and VPA on the DR3 and analysis of the methylation status on the DR3 gene promoter in OS cell lines. U‑2 OS and SaOS‑2 cells were treated with 20 µM Hy and/or 1.0 mM VPA for 3 (Day 3) or 7 days (Day 7). The medium was changed on day 3. (A) DR3 mRNAs was quantitated using qPCR analysis. The values are expressed as the ratio to the average value in the no treat as a control. Each bar indicates the mean ± SE of values from four independent experiments for each sample, in three sets of culture conditions. *P<0.01, significant difference in comparison to no treat and to Hy. **P<0.05, significant difference in comparison to Hy. (B) The DR3 protein expression was examined by western blotting. (C) Methylation‑specific PCR (MSP) was performed with 20 µM Hy and/or 1.0 mM VPA treatment on day 3 in SaOS‑2 cells. Bisulfited genomic DNA amplified by unmethylated‑specific (U) and methylated‑specific (Me) primers designed in the CpG island of the DR3 promoter region. M, molecular marker. Hy, hydralazine; VPA, sodium valproate; DR3, death receptor‑3; OS, osteosarcoma; qPCR, quantitative polymerase chain reaction; SE, standard error.

Article Snippet: The membrane was blocked for 30 min at room temperature in blocking buffer containing 5% skimmed milk in Tris-buffered saline with Tween-20 [TBST; 10 mM Tris, 150 mM NaCl (pH 7.4), 1% Tween-20] and then incubated for 90 min at room temperature with anti-human VEGI mouse monoclonal antibody (1:200 dilution; Santa Cruz Biotechnology, Inc., cat. no. sc-53975) or anti-human DR3 mouse monoclonal antibody (1:500 dilution; Santa Cruz Biotechnology, Inc.; cat. no. sc-374203) in TBST buffer, after which time the membrane was washed with TBST three times (10 min each time).

Techniques: Methylation, Control, Comparison, Expressing, Western Blot, Amplification, Marker, Real-time Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: Epigenetic modulators hydralazine and sodium valproate act synergistically in VEGI-mediated anti-angiogenesis and VEGF interference in human osteosarcoma and vascular endothelial cells.

doi: 10.3892/ijo.2019.4811

Figure Lengend Snippet: Figure 4. Effects of Hy and VPA on VEGI and its related receptors on transcription and the production of their soluble forms in the HMVE cells. HMVE cells were cultured in medium with 20 µM Hy and/or 1.0 mM VPA for 7 days (Day 7). The medium was changed on day 3. The gene expression levels of (A) VEGI, (C) DR3 and (E) DcR3 were quantitated using qPCR. The values are expressed as the ratio to the mean value in the no treat as a control. Each bar indicates the mean ± SE of values obtained from four independent experiments for each sample, under three sets of culture conditions. *P<0.01, significant difference in comparison to no treat and to Hy. **P<0.05, significant difference in comparison to Hy. The (B) VEGI and (D) DR3 protein expression levels were examined using western blotting. The amount of (F) soluble DcR3 and (G) VEGI in the medium was determined on Day 7. The values are indicated as the ratio of the mean amounts of soluble DcR3 and VEGI per 104 viable cells in no treat is expressed as 1.0. Each bar indicates the mean ± SE of values obtained from four independent experiments for each sample, under three sets of culture conditions. *P<0.001, significant difference in comparison to no treat and to Hy. Hy, hydralazine; VPA, sodium valproate; VEGI, vascular endothelial growth inhibitor; HMVE, human microvascular endothelial; DR3, death receptor‑3; DcR3, decoy receptor‑3; qPCR, quantitative polymerase chain reaction; SE, standard error.

Article Snippet: The membrane was blocked for 30 min at room temperature in blocking buffer containing 5% skimmed milk in Tris-buffered saline with Tween-20 [TBST; 10 mM Tris, 150 mM NaCl (pH 7.4), 1% Tween-20] and then incubated for 90 min at room temperature with anti-human VEGI mouse monoclonal antibody (1:200 dilution; Santa Cruz Biotechnology, Inc., cat. no. sc-53975) or anti-human DR3 mouse monoclonal antibody (1:500 dilution; Santa Cruz Biotechnology, Inc.; cat. no. sc-374203) in TBST buffer, after which time the membrane was washed with TBST three times (10 min each time).

Techniques: Cell Culture, Gene Expression, Control, Comparison, Expressing, Western Blot, Real-time Polymerase Chain Reaction