Human/Mouse/Rat TRAF-2 Antibody

Detection of Human/Mouse/Rat TRAF‑2 by Western Blot. Western blot shows lysates of Raji human Burkitt's lymphoma cell line, SW3T3 mouse contact inhibited fibroblast cell line, and L6 rat myoblast cell line. PVDF membrane was probed with 0.25 µg/mL Mouse Anti-Human/Mouse/Rat TRAF-2 Monoclonal Antibody (Catalog # MAB3277) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF007). For additional reference, recombinant human TRAF-1, -2, -3, -4, -5, and -6 (2 ng/lane) were included. A specific band for TRAF-2 was detected at approximately 56 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 2.

TRAF‑2 in HeLa Human Cell Line. TRAF-2 was detected in immersion fixed HeLa human cervical epithelial carcinoma cell line using Mouse Anti-Human/Mouse/Rat TRAF-2 Monoclonal Antibody (Catalog # MAB3277) at 3 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Mouse IgG Secondary Antibody (red; Catalog # NL007) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Western Blot Shows Human TRAF‑2 Specificity by Using Knockout Cell Line. Western blot shows lysates of HEK293T human embryonic kidney parental cell line and TRAF-2 knockout HEK293T cell line (KO). PVDF membrane was probed with 0.25 µg/mL of Mouse Anti-Human/Mouse/Rat TRAF-2 Monoclonal Antibody (Catalog # MAB3277) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). A specific band was detected for TRAF-2 at approximately 62 kDa (as indicated) in the parental HEK293T cell line, but is not detectable in knockout HEK293T cell line. GAPDH (Catalog # MAB5718) is shown as a loading control. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of TRAF-2 by Western Blot RAR gamma is required for TNF-induced RIP1-dependent apoptosis and necroptosis. a Cell death analysis of HT-29 clones cont-shRNA, RAR gamma -shRNA-A, RAR gamma -shRNA-B, RAR gamma -shRNA-C or RIP1-shRNA when treated with TSZ for 24 h was determined by PI staining using flow cytometry (left panel) (*P < 0.05 versus cont-shRNA; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel; cell lysates were probed with antibodies as indicated (right panel). b Cell death determination by PI staining using flow cytometry of HT-29 cont-shRNA, RAR gamma -shRNA-A or RAR gamma -shRNA-A cells reconstituted with RAR gamma -shRNA resistant RAR gamma protein (RAR gamma -shRNA-A-rRAR gamma ) when treated with DMSO or TSZ for 24 h (left panel) (*P < 0.05 versus cont-shRNA. #P < 0.05 versus RAR gamma -shRNA-A; ANOVA). The bars represent the mean ± s.e.m. of three experiments. Western blot analysis of cells as mentioned in left panel; cell lysates were probed with antibodies as indicated (right panel). c, d HT-29, RAR gamma -shRNA-A, or RIP1-shRNA cells were treated with necrotic (DMSO, TS, or TSZ) c or apoptotic (CHX, TC or TCZ) d conditions for 24 h. PI positive population was determined by flow cytometry. (*P < 0.05 versus cont-shRNA; ANOVA). The bars represent the mean ± s.e.m. of three experiments. e Western blot analysis of HT-29 cont-shRNA cells infected with TRADD-shRNA, or RAR gamma -shRNA-A cells infected with TRADD-shRNA lentivirus (RAR gamma -shRNA-A + TRADD-shRNA). Cell lysates were probed with antibodies as indicated. f Cells from e were treated with DMSO, TS, or TSZ for 24 h and the PI-positive population was determined by flow cytometry. (*P < 0.05 versus cont-shRNA; ANOVA). The bars represent the mean ± s.e.m. of three experiments. All blots above are representative of one of three experiments Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28871172), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of TRAF-2 by Western Blot Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of TRAF-2 by Western Blot Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of TRAF-2 by Western Blot Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of TRAF-2 by Western Blot Analysis of the activity of CD40 related proteins involved in transduction leading to activation of key inflammatory/survival pathway and transcription factors. (A, B) Immature dendritic cells were differentiated from monocytes derived from PBMCs donors and treated with the indicated treatments over 4 hours. (A) Representative western blot analysis (n=3-6) of CD40 related signaling adaptor proteins and related inflammatory/survival pathway and transcription factors using beta-actin as a loading control. HERA-CD40L treatment is highlighted by purple rectangles shaded by timepoints. (B) Representative subcellular fractionation and western blot analysis 15 minutes after treatment (n=3), the indicated subcellular fractions are determined by cytosolic, RIPA soluble and insoluble samples, as indicated. RIPA samples correspond to membrane and nuclear fractions while RIPA insoluble samples correspond to insoluble lipid rafts, nuclear insoluble fraction, and bound nuclear chromatin. (C) Dendritic cell activation surface marker expression analysis and IL-12 production comparison between the indicated treatments. Data displayed as mean +/- SEM (n=3 donors), Standard unpaired t-test comparison. Statistics are represented as either not significant (ns), p-value = <0.05 (*), or p-value = <0.005 (***). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37304285), licensed under a CC-BY license. Not internally tested by R&D Systems.