Afuco™ Anti-Human TNFRSF21 ADCC Recombinant Antibody (DQM3), ADCC Enhanced (CAT#: AFC-607CL)

Anti-TNFRSF21 ADCC Enhanced Antibody (DQM3) is an ADCC enhanced antibody produced by our Afuco™ platform. DQM3 is a functional agonist monoclonal antibody against DR6, which bound to the first cysteine-rich domain. Death receptor 6 (DR6) is a member of the death domain-containing receptors that belong to the TNFR superfamily.


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FC

Figure 1 Mapping binding epitopes of anti-DR6 mAbs.

Figure 1 Mapping binding epitopes of anti-DR6 mAbs.

(A) HEK293T cells were transiently transfected with empty vector or expression vector encoding DR6-FL. Transfected cells were indicated by the expression of GFP. DR6 expression was analyzed by flow cytometry with anti-DR6 mAbs (DQM2, DQM3 and DQM5) or isotype control antibodies. EV, empty vector; black line, anti-DR6 mAbs; grey fill, isotype control antibodies. (B) Schematic representation of the DR6 deletion mutants used in this study (intracellular domain is not shown). The tested DR6 deletion mutants targeting extracellular domain include DCRD3-4, DCRD1-2, DCRD2, DCRD1, D67-88 and D50-66. (C) DR6 and its mutants were transfected into HEK293T cells. Cells were collected after 36 h, and FACS analysis was performed to detect the binding of DQM2, DQM3 and DQM5.

Hu, R., Du, Q., Yin, X., Li, J., Wang, T., & Zhang, L. (2014). Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment. FEBS letters, 588(3), 401-407.

Stim

Figure 2 DQM3 stimulates DR6-induced IL-6 luciferase activity.

Figure 2 DQM3 stimulates DR6-induced IL-6 luciferase activity.

(A) HEK293T cells were co-transfected with DR6-FL, IL-6 luciferase reporter and Renilla. Anti-DR6 mAbs (DQM2, DQM3 and DQM5, 10 lg/ml) were added 24 h after transfection, and isotype control antibody alone also served as a negative control. Luciferase assays were performed to measure promoter activity after 16 h of antibody incubation. (B) Transfected cells in (A) were stimulated with increasing amounts of DQM3. Luciferase activity was determined as described in (A). (C) HEK293T cells were transfected with increasing amounts of vectors for DR6-FL or DR6-EX. 36 h later, cell lysates were prepared and subjected to Western blot analysis using antibodies to phospho-JNK, total JNK and phospho-IjBa. b-Actin was used as loading control. (D) Cells transfected with DR6-FL in (C) were left untreated or treated with DQM3 (10 lg/ml) for the indicated times. Western blot analysis was performed as described in (C).

Hu, R., Du, Q., Yin, X., Li, J., Wang, T., & Zhang, L. (2014). Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment. FEBS letters, 588(3), 401-407.

Figure 3 Death domain is essential for DR6-induced signaling upon DQM3 stimulation.

Figure 3 Death domain is essential for DR6-induced signaling upon DQM3 stimulation.

(A) Schematic representation of DR6 mutants used in this study. The deletion mutants targeting intracellular domain include DR6-EX (1-370), DR6 (1-414), DR6 (1-498), DR6-DTRAF (D371-414) and DR6-DDD (D415-498). (B) HEK293T cells were transfected with IL-6 reporter plasmid alone or together with a series of expression vectors encoding DR6 or its mutants as indicated above. 24 h later, cells were stimulated with DQM3 or isotype control antibody. Cell lysates were prepared 16 h after antibody incubation and relative luciferase activity of stimulus versus control was calculated as the mean ± S.E.M.

Hu, R., Du, Q., Yin, X., Li, J., Wang, T., & Zhang, L. (2014). Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment. FEBS letters, 588(3), 401-407.

Figure 4 TRADD is involved in DR6-induced IL-6 activation.

Figure 4 TRADD is involved in DR6-induced IL-6 activation.

(A and B) HEK293T cells were transiently transfected with IL-6 reporter plasmid, Renilla and increasing amounts of expression vectors for TRADD or TRADD-DN (A) or together with DR6-FL (B). 24 h later, promoter activity was measured by luciferase assay. (C) HEK293T cells were cotransfeced with IL-6-Luc, Renilla, DR6-FL and TRADD as described in (B), and then stimulated with DQM3. 16 h later, luciferase activity was measured. (D) HEK293T cells were co-transfected with TRADD siRNA (si-TRADD) or negative control siRNA (si-NC) and expression plasmid for TRADD. Western blot analysis was performed to verify the efficiency of siRNA. (E) TRADD siRNA-treated cells were transfected with IL-6 reporter plasmid and DR6-FL. 24 h after transfection, cells were subjected to DQM3 stimulation for 16 h, and then luciferase activity was monitored as described above.

Hu, R., Du, Q., Yin, X., Li, J., Wang, T., & Zhang, L. (2014). Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment. FEBS letters, 588(3), 401-407.

IP

Figure 5 DR6 interacts with TRADD in co-immunoprecipitation assays.

Figure 5 DR6 interacts with TRADD in co-immunoprecipitation assays.

(A and B) HEK293T cells in 60-mm dishes co-transfected with DR6-FL/EX and Flag-tagged TRADD or TRADD-DN were harvested 48 h after transfection. Total cell lysates were immunoprecipitated with anti-DR6 (DQM3) and immunoblotted with anti-TRADD (A) or anti-Flag (B). Western blot analysis of DR6, TRADD and Flag was performed on whole cell lysates as a control (Input). (C) HEK293T cells were co-transfected with expression vectors for DR6-FL and Flag-tagged TRADD, and then stimulated or not with DQM3 for the indicated times. The interaction of TRADD with DR6 was assessed after DQM3 stimulation by co-immunoprecipitation. The immunoprecipitates and cell extracts were analyzed by immunoblot.

Hu, R., Du, Q., Yin, X., Li, J., Wang, T., & Zhang, L. (2014). Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment. FEBS letters, 588(3), 401-407.


Specifications

  • Host Species
  • Human
  • Type
  • ADCC enhanced antibody
  • Species Reactivity
  • Human
  • Applications
  • FC, Stim, IP
  • Related Disease
  • Cancer

Product Property

  • Purity
  • >95%, by SDS-PAGE with Coomassie Brilliant Blue staining
  • Storage
  • Short term: store at 4°C (over 6 months), long term: -20°C or -80°C.

Applications

  • Application Notes
  • The antibody was validated for Flow Cytometry, Stimulation, Immunoprecipitation. For details, refer to Published Data.

Target

  • Alternative Names
  • TNFRSF21; tumor necrosis factor receptor superfamily, member 21; DR6; CD358; BM-018; tumor necrosis factor receptor superfamily member 21; death receptor 6; TNFR-related death receptor 6

Product Notes

This is a product of Creative Biolabs' Hi-Affi™ recombinant antibody portfolio, which has several benefits including:

• Increased sensitivity
• Confirmed specificity
• High repeatability
• Excellent batch-to-batch consistency
• Sustainable supply
• Animal-free production

See more details about Hi-Affi™ recombinant antibody benefits.

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Rat Antibody

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TAB-631CL Anti-Human TNFRSF21 Recombinant Antibody (DQM3) Stim Antibody

Mouse Antibody

Human Antibody

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.

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