Anti-DR3/Tnfrsf25 Antibody Picoband®

Western blot analysis of DR3/Tnfrsf25 using anti-DR3/Tnfrsf25 antibody (A03227-3).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: mouse SP2/0 whole cell lysates,
Lane 2: mouse RAW264.7 whole cell lysates,
Lane 3: mouse NIH/3T3 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-DR3/Tnfrsf25 antigen affinity purified polyclonal antibody (Catalog # A03227-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for DR3/Tnfrsf25 at approximately 60KD. The expected band size for DR3/Tnfrsf25 is at 60KD.

Methylating effect of daphnetin and 5-aza-dc on DR3, PDCD5, FasL and p53 in CIA rat synovial cells. Four experimental groups consisting of untreated cell control, or treated with 5-aza-dc at 20 μM, daphnetin at 40 μg/mL, or combination of 5-aza-dc (20 μM) and daphnetin (40 μg/mL). Cells were cultured, treated and harvested as described in Materials and Methods. Total DNA was extracted and used for MSP after bisulphite modification. M: methylated amplification products, U: hypomethylated amplification products.
Index in PubMed under a CC BY license. PMID: 25311560

Effect of daphnetin and 5-aza-dc on expression of DR3, PDCD5, FasL, p53 (A) and DNMT1, DNMT3a, DNMT3b (B) in CIA rat synovial cells. Four experimental groups consisting of untreated cell control, or treated with 5-aza-dc at 20 μM, daphnetin at 40 μg/mL, or combination of 5-aza-dc (20 μM) and daphnetin (40 μg/mL). Cells were cultured, treated and harvested as described in Matreials and Methods. Total RNA was extracted and cDNA was synthesized. After reverse transcription, cDNA was used for real time-PCR. Relative quantification of gene expression was performed by the 2-ΔΔCt method. The results show the mean ± S.D. of six independent experiments. ▲P < 0.05 compared with control, ●P < 0.05 compared with 5-aza-dc, ☆P > 0.05 compared with 5-aza-dc, ★P > 0.05 compared with daphnetin, *P < 0.05 compared with daphnetin.
Index in PubMed under a CC BY license. PMID: 25311560

Effect of daphnetin on DR3, PDCD5, FasL and p53 protein expression in CIA rats synovial cells. Cells were cultured, treated and harvested as described Materials and Methods. Four experimental groups consisting of untreated cell control, 5-aza-dc at 20 μM, daphnetin at 40 μg/mL, or combination of 5-aza-dc (20 μM) and daphnetin (40 μg/mL). The results show the mean ± S.D. of six independent experiments. ▲P < 0.05 compared with control, ●P < 0.05 compared with 5-aza-dc, ★P > 0.05 compared with 5-aza-dc, *P < 0.05 compared with daphnetin.
Index in PubMed under a CC BY license. PMID: 25311560

Flow Cytometry analysis of NRK cells using anti-DR3/Tnfrsf25 antibody (A03227-3).
Overlay histogram showing NRK cells stained with A03227-3 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-DR3/Tnfrsf25 Antibody (A03227-3, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.