Human BAFF/BLyS/TNFSF13B Antibody

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.