Human TNF Receptor II protein, His tag (active)

Tumor necrosis factor receptor superfamily member 1B (TNFRSF1B), also known as tumor necrosis factor receptor 2 (TNFR2) and CD120b, is a membrane receptor that binds tumor necrosis factor-alpha (TNFα). This protein and TNF-receptor 1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. Besides, Tumor Necrosis Factor Alpha (TNFa) has been identified as an interactor of TNFRSF1B, thus a binding ELISA assay was conducted to detect the interaction of recombinant human TNFRSF1B and recombinant human TNFa. Briefly, TNFRSF1B were diluted serially in PBS, with 0. 01% BSA (pH 7. 4). Duplicate samples of 100 μl were then transferred to TNFa-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-TNFRSF1B pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of TNFRSF1B and TNFa was in a dose dependent manner.
To tested cell apoptosis, A549 cells were seeded into triplicate wells of 96-well plates at a density of 2000 cells/well. and allowed to attach, replaced with serum-free overnight, then the medium was replaced with 2% serum standard DMEM including 1μg/ml TNFα and various concentrations of recombinant human TNFRSF1B. After incubated for 96h, cells were observed by inverted microscope and cell proliferation was measured by Cell Counting Kit-8 (CCK-8). Briefly, 10 μl of CCK-8 solution was added to each well of the plate, then the absorbance at 450nm was measured using a microplate reader after incubating the plate at 37ºC for 1-4 hours. Apoptosis of A549 cells had been inhibit after incubation with TNFRSF1B for 96h observed by inverted microscope . Cell viability was assessed by CCK-8 (Cell Counting Kit-8) assay after incubation with recombinant TNFRSF1B for 96h. And TNFRSF1B significantly suppress cell apoptosis induced by TNFα.