Recombinant Mouse Cd40 protein, His-tagged

• Cat.No.: Cd40-81M
• Product Overview: Recombinant mouse CD40, fused to His-tag at C-terminus, was expressed in insect cell and purified by using conventional chromatography techniques.

Specification

• Species: Mouse
• Source: Insect Cells
• Tag: His
• Description: CD40, also known as tumor necrosis factor receptor superfamily member, is receptor for TNFSF5/CD40LG. It transduces TRAF6 and MAP3K8 mediated signals that activate ERK in macrophages and B cells, leading to induction of immunoglobulin secretion.
• Form: Liquid. In Phosphate Buffered Saline (pH 7.4) containing 10% glycerol.
• Molecular Mass: 20.1kDa (180aa)18-28KDa (SDS–PAGE under reducing conditions.)
• AA Sequence: LGQCVTCSDK QYLHDGQCCD LCQPGSRLTS HCTALEKTQC HPCDSGEFSA QWNREIRCHQ HRHCEPNQGL RVKKEGTAES DTVCTCKEGQ HCTSKDCEAC AQHTPCIPGF GVMEMATETT DTVCHPCPVG FFSNQSSLFE KCYPWTSCED KNLEVLQKGT SQTNVICGLK SRMRHHHHHH
• Endotoxin: < 1.0 EU per 1 microgram of protein (determined by LAL method)
• Purity: : >95% by SDS – PAGE
• Storage: Can be stored at +4 centigrade short term (1-2 weeks). For long term storage, aliquot and store at -20 centigrade or -70 centigrade. Avoid repeated freezing and thawing cycles.
• Concentration: 1.0mg/ml (determined by Absorbance at 280nm)

Gene Information

• Gene Name: Cd40 CD40 antigen [ Mus musculus ]
• Official Symbol: Cd40
• Synonyms: CD40; CD40 antigen; tumor necrosis factor receptor superfamily member 5; CD40L receptor; B-cell surface antigen CD40; T-cell differentiation antigen; tumor necrosis factor receptor superfamily, member 5; IGM; p50; Bp50; GP39; IMD3; TRAP; HIGM1; T-BAM; Tnfrsf5; AI326936
• Gene ID: 21939
• mRNA Refseq: NM_011611
• Protein Refseq: NP_035741
• Pathway: Adaptive Immune System, organism-specific biosystem; Allograft rejection, organism-specific biosystem; Allograft rejection, conserved biosystem; Asthma, organism-specific biosystem; Asthma, conserved biosystem; Autoimmune thyroid disease, organism-specific biosystem; Autoimmune thyroid disease, conserved biosystem
• Function: binding; enzyme binding; protein binding; receptor activity

Citation

TREM2 deficiency reduces the efficacy of immunotherapeutic amyloid clearance

Journal: EMBO Molecular Medicine    PubMed ID: 27402340    Data: 2016/7/8

Authors: Xianyuan Xiang, Georg Werner, Christian Haass

Article Snippet:Before measurement, medium was removed and extracellular fAβ 42 was quenched with 100 μl 0.2% trypan blue in phosphate‐buffered saline (PBS), pH 4.4 for 1 min. After aspiration, fluorescence signals were measured at 485‐nm excitation/538‐nm emission using a Fluoroskan Ascent ? Microplate Fluorometer (Lab Systems).Before measurement, medium was removed and extracellular fAβ 42 was quenched with 100 μl 0.2% trypan blue in phosphate‐buffered saline (PBS), pH 4.4 for 1 min. After aspiration, fluorescence signals were measured at 485‐nm excitation/538‐nm emission using a Fluoroskan Ascent ? Microplate Fluorometer (Lab Systems).. For the experiment shown in Fig I, BMDM were incubated with recombinant mouse sTREM2 (Creative BioMart) overnight before adding fAβ 42 or antibody‐fAβ 42 complexes.

Schematic of the mouse Trem2 locus and the TREM2 protein. Sequence alignment of wild‐type N9 (N9 wt) and TREM2 mutant N9 (N9 mu) surrounding the gRNA target site. The gRNA sequence is in cyan, and protospacer‐adjacent motif (PAM) is marked with a line. The single nucleotide insertion is labeled in red. Schematic representation of wild‐type TREM2 (NP_112544.1) and CRISPR/Cas9‐modified TREM2 (N9 mu). TM, transmembrane domain; SP, signal peptide. Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using the antibody anti‐murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2. *indicate unspecific bands. Calnexin was used as a loading control. Phagocytosis of 1 μM HiLyte ? Fluor 488 Aβ 1‐42 (fAβ 42 ) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non‐binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. ( n = 4, ± SEM; two‐way ANOVA, interaction P = 0.61, genotype P < 0.0001, treatment P = 0.0001; post hoc tests wt vs. mu for the following conditions: fAβ 42 P = 0.0043, fAβ 42 ‐2D8 P = 0.0436). Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4. *indicate unspecific bands. Phagocytosis of fAβ 42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non‐binding control antibody 6687. ( n = 3, ± SEM; two‐way ANOVA, interaction P = 0.0005, genotype P < 0.0001, treatment P < 0.0001; post hoc tests wt vs. ko for the following conditions: fAβ 42 P = 0.0021, fAβ 42 ‐2D8 1 μg/ml P < 0.0001, fAβ 42 ‐2D8 5 μg/ml P < 0.0001, fAβ 42 ‐2D8 10 μg/ml P < 0.0001, fAβ 42 /6687 10 μg/ml P = 0.0007). Quantification of relative fAβ 42 uptake to lowest antibody concentration used ( n = 3, ± SEM). Phagocytosis of fAβ 42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). ( n = 4, ± SEM; two‐way ANOVA, interaction P = 0.0223, genotype P < 0.0001, treatment P < 0.0001; post hoc tests wt vs. ko for the following conditions: fAβ 42 ‐mAb11 1 μg/ml P = 0.0391, fAβ 42 ‐mAb11 5 μg/ml P = 0.0069, fAβ 42 ‐mAb11 10 μg/ml P < 0.0001, fAβ 42 ‐mAb11 20 μg/ml P = 0.0001, fAβ 42 ‐mAb11 50 μg/ml P < 0.0001). Quantification of relative fAβ 42 uptake to lowest antibody concentration used ( n = 4, ± SEM). Recombinant mouse sTREM2 does not rescue fAβ 42 uptake in Trem2 ‐deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 μg/ml) ( n = 4, ± SEM). Western blot of primary microglia from wt or Trem2 ko animals using antibody 5F4. *indicate unspecific bands. Phagocytosis of fAβ 42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). ( n = 5, ± SEM; two‐way ANOVA, interaction P = 0.4797, genotype P < 0.0001, treatment P < 0.0001; post hoc tests wt vs. ko for the following conditions: fAβ 42 ‐mAb11 5 μg/ml P = 0.0449, fAβ 42 ‐mAb11 10 μg/ml P = 0.0370, fAβ 42 ‐mAb11 20 μg/ml P = 0.0299, fAβ 42 ‐mAb11 50 μg/ml P = 0.0120). Quantification of relative fAβ 42 uptake to lowest antibody concentration used ( n = 5, ± SEM). Data information: (C, E, G, K) Quantification of internalized fAβ 42 was normalized to wt without antibody. Bonferroni‐corrected pair‐wise post hoc tests were used. Source data are available online for this figure.