Human BAFF/BLyS/TNFSF13B Antibody

Cell Proliferation Induced by BAFF/BLyS/TNFSF13B and Neutralization by Human BAFF/BLyS/TNFSF13B Antibody. Recombinant Human BAFF/BLyS/TNFSF13B (Catalog # 2149-BF) stimulates proliferation in mouse B cells in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Human BAFF/BLyS/TNFSF13B (5 ng/mL) is neutralized (green line) by increasing concentrations of Mouse Anti-Human BAFF/BLyS/TNFSF13B Monoclonal Antibody (Catalog # MAB124). At 20 µg/mL, this anti-BAFF/BLyS/TNFSF13B antibody will neutralize >60% of Recombinant Human BAFF/BLyS/TNFSF13B-induced proliferation of mouse B cells.

Detection of BAFF/BLyS/TNFSF13B by Western Blot BAFF-mediated signaling synergizes with LPS for NF-kappa B activation without affecting I kappa B degradation or NF-kappa B nuclear translocation.(A and B) HEK 293 T cells were transfected with an NF-kappa B luciferase construct, reference reporter construct, and BAFF expression vector in combination with CD4-TLR4, death domain (DD) of MyD88, or wild type TRIF-expressing vectors for 24 h. Relative luciferase activity (RLA) was then measured. ***p < 0.001 when compared with the negative control. #p < 0.05, ## < 0.01 and ### < 0.001 (n = 6). (C) THP-1 cells were pre-treated with 1 μg/ml of anti-BAFF mAb or mIgG and then stimulated with 100 ng/ml of LPS for the indicated time periods and total cell lysates were obtained. The Western blot was performed using antibodies specific for phospho-I kappa B-alpha, I kappa B-alpha or beta -actin. (D) Cells were pretreated as in C and stimulated with 1 μg/ml of LPS. After 30 min, the location of p65 was analyzed using immunocytochemistry and the proportion of cells with nuclear p65 was determined (n = 3). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28374824), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAFF/BLyS/TNFSF13B by Flow Cytometry The enhancing effect of mAb is BAFF specific and starts at relatively early time points after LPS stimulation.(A) THP-1 cells were transiently transfected with 10 nM scramble siRNA (NC) or BAFF-specific siRNA. After 24 h, RT-PCR analysis was performed to evaluate the expression levels of BAFF mRNA. (B) Cells were transfected as in (A) and collected at 48 h. BAFF expression levels were then measured by flow cytometry using BAFF specific mAb (empty area) or mIgG (filled area). (C) Cells were transfected as in (A). After 48 h, cells were pre-treated with 1 μg/ml mIgG or anti-BAFF mAb for 30 min and then the cells were stimulated with 100 ng/ml LPS for 24 h. Cultured supernatants were then collected to evaluate levels of secreted IL-8. Values represent IL-8 levels relative to those of the positive control (set to 100%). *p < 0.05 (n = 3). (D) THP-1 cells were pretreated as in (C) and stimulated with 1 μg/ml of LPS. Culture supernatants were collected at the indicated time points and IL-8 levels were measured using ELISA. Control samples were treated with 1 μg/ml anti-BAFF or mIgG for 24 h in the absence of LPS treatment. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28374824), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAFF/BLyS/TNFSF13B by Western Blot The PI3K/AKT/CREB pathway contributes to the synergistic activation of NF-kappa B and expression of IL-8.(A) THP-1 cells were pre-treated with anti-BAFF or mIgG (1 μg/ml) for 30 min and then stimulated with 100 ng/ml of LPS for the indicated times. Total cell lysates were tested for the levels of phospho-AKT(Ser473), AKT, and beta -actin by Western blot. (B) THP-1 cells were pre-treated with 5 μM LY294002 (PI3K inhibitor), 10 μM MK2206 (AKT inhibitor) or 0.05% DMSO (VC) for 1 h. The cells were treated with anti-BAFF (1 μg/ml) for 30 min and then stimulated with LPS (100 ng/ml) for 24 h. The levels of secreted IL-8 in culture supernatants were determined by ELISA. **p < 0.01 when compared with corresponding positive control samples stimulated in the absence of inhibitors (n = 6). (C) HEK 293 T cells were co-transfected with an NF-kappa B luciferase construct, reference reporter construct, and BAFF and CD4-TLR4 expressing vectors. After 3 h, the cells were treated with 20 μM LY294002 (PI3K inhibitor), 10 μM MK2206 (AKT inhibitor) or 0.2% DMSO (VC). After 21 h, relative luciferase activities (RLA) were measured. **p < 0.01 when compared with cells co-transfected with BAFF and CD4-TLR4 expressing vectors (n = 6). (D) THP-1 cells were pre-treated with 20 μM LY294002 (PI3K inhibitor) or 0.2% DMSO (VC) for 1 h. The cells were stimulated with LPS (100 ng/ml) for indicated times in the presence of anti-BAFF mAb (1 μg/ml). The phosphorylation levels of AKT and CREB were analyzed by Western blot. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28374824), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAFF/BLyS/TNFSF13B by Western Blot BAFF exerts its enhancing effects through activation of ERK and JNK MAPKs.(A) THP-1 cells were stimulated with anti-BAFF (1 μg/ml) mAb for the indicated time periods. The activation status of MAPK was analyzed by Western blot using antibodies specific for normal and phosphorylated form of MAPKs and actin. (B) The cells were pre-treated with 1 μg/ml of anti-BAFF mAb or mIgG for 30 min and then stimulated with 100 ng/ml of LPS for the indicated time periods. The Western blot analysis of MAPKs was performed as in A. (C-F) THP-1 cells were pre-treated with 3.75 μM U0126 (ERK inhibitor), 2 μM SB203580 (p38 inhibitor), 10 μM SP600125 (JNK inhibitor) or 0.02% DMSO (VC) for 30 min. Cells were then treated with 1 μg/ml anti-BAFF for 30 min before stimulation with 100 ng/ml of LPS for 24 h. Culture supernatants were collected and levels of secreted MMP-9 (C), IL-8 (D), TNF-alpha (E) and MCP-1 (F) were analyzed by gelatin zymography and ELISA. *p < 0.05 when compared with corresponding positive control samples stimulated in the absence of inhibitors (n = 3). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28374824), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAFF/BLyS/TNFSF13B by Western Blot BAFF exerts its enhancing effects through activation of ERK and JNK MAPKs.(A) THP-1 cells were stimulated with anti-BAFF (1 μg/ml) mAb for the indicated time periods. The activation status of MAPK was analyzed by Western blot using antibodies specific for normal and phosphorylated form of MAPKs and actin. (B) The cells were pre-treated with 1 μg/ml of anti-BAFF mAb or mIgG for 30 min and then stimulated with 100 ng/ml of LPS for the indicated time periods. The Western blot analysis of MAPKs was performed as in A. (C-F) THP-1 cells were pre-treated with 3.75 μM U0126 (ERK inhibitor), 2 μM SB203580 (p38 inhibitor), 10 μM SP600125 (JNK inhibitor) or 0.02% DMSO (VC) for 30 min. Cells were then treated with 1 μg/ml anti-BAFF for 30 min before stimulation with 100 ng/ml of LPS for 24 h. Culture supernatants were collected and levels of secreted MMP-9 (C), IL-8 (D), TNF-alpha (E) and MCP-1 (F) were analyzed by gelatin zymography and ELISA. *p < 0.05 when compared with corresponding positive control samples stimulated in the absence of inhibitors (n = 3). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28374824), licensed under a CC-BY license. Not internally tested by R&D Systems.