cd86 fc  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: Huanshaodan regulates microglial glucose metabolism reprogramming to alleviate neuroinflammation in AD mice through mTOR/HIF-1α signaling pathway

doi: 10.3389/fphar.2024.1434568

Figure Lengend Snippet: Expression levels of CD86, Arg1 and IL-1β protein in hippocampus and cortex of SAMP8 mice detected by Western-blot, the expression levels of IL-6 and TNF-α in hippocampus and cortex of SAMP8 mice by ELISA. (A,E) Blotting of CD86, Arg1 and IL-1β protein in hippocampus and cortex of mice in each group; (B–D) Expression of CD86, Arg1 and IL-1β protein in hippocampus of mice in each group; (F–H) Expression of CD86, Arg1 and IL-1β protein in cortex of mice in each group; (I–L) Expression of IL-6 and TNF-α protein in hippocampus and cortex of mice in each group. Model group compared with the control group * p < 0.05, ** p < 0.01, *** p < 0.001, Donepezil group compared with the model group # p < 0.05, ## p < 0.01, ### p < 0.001, HSD group compared with the model group $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

Article Snippet: The main materials that were used in this study were LPS (L8880, Solarbio), MHY1485 (MHY) (M9050-5MG, AbMole), RIPA buffer (high) (R0010, Solarbio), primary antibodies β-Actin and CD86 (42kDa, BM0627; 70kDa, BM4121, BOSTER), Arg1 and mTOR (40kDa, GB11285-100; 289kDa, GB111839-100, Servicebio), p-mTOR and HIF-1α (289kDa, 5536T; 120kDa, 48085S, Cell Signaling Technology), IL-1β (31kDa, 16806-1-AP, Proteintech), Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies (ZB-2301; ZB-2305, Beijing Zhong Shan-Golden Bridge Biological Technology), Glucose (GLU) Assay Kit, Lactic Acid (LD) assay kit, ATP assay kit, Lactate dehydrogenase (LDH) assay kit (A154-1-1; A019-2-1; A095-1-1; A020-2-2, Nanjing Jiancheng Bioengineering Institute), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (E-BC-K650-M, Elabscience), BCA Protein Assay Kit (PC0020, Solarbio), ELISA Kit for Hexokinase 2 (HK2) activity based on enzyme standard of HK2, ELISA Kit for Tumor Necrosis Factor alpha (TNF-α) (MM-45418M1; MM-0132M1, MEIMIAN), ELISA Kit for Interleukin 6 (IL-6) (E-MSEL-M0001, Elabscience) and ELISA Kit for Pyruvate kinase isozymes M2 (PKM2) (SEA588Mu, Cloud Clone Corp), Cell Staining Buffer, PE Anti-Mouse F4/80 Antibody [CI:A3-1], PE/Cyanine7 Anti-Mouse CD86 Antibody [GL-1], Intracellular Fixation/Permeabilization Buffer Kit and APC Anti-Mouse CD206 Antibody (E-CK-A107; E-AB-F0995D; E-AB-F0994H; E-CK-A109; E-AB-F1135E, Elabscience).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Huanshaodan regulates microglial glucose metabolism reprogramming to alleviate neuroinflammation in AD mice through mTOR/HIF-1α signaling pathway

doi: 10.3389/fphar.2024.1434568

Figure Lengend Snippet: The expression levels of p-mTOR/mTOR ratio, HIF-1α, and IL-1β in BV2 cells tested by western blot, the levels of GLU, LD, ATP, LDH and PDH quantified by biochemical test kit and the levels of polarization biomarkers of CD86 and CD206 detected by flow cytometry, the expression levels of IL-6 and TNF-α in BV2 cells tested by ELISA. (A) Blotting of p-mTOR, mTOR and HIF-1α protein in BV2 cells in each group; (B, C) Expression levels of p-mTOR/mTOR ratio and HIF-1α in BV2 cells in each group. (D) GLU content analysis chart; (E) LD content analysis chart; (F) ATP content analysis chart; (G) . LDH activity analysis graph; (H) PDH activity analysis graph. (I) Flow cytometry analysis charts; (J) Flow cytograms of BV2 cells in each group; (K) Blotting of IL-1β protein in BV2 cells in each group; (L) Expression level of IL-1β in BV2 cells in each group. (M, N) Expression level of IL-6 and TNF-α in BV2 cells in each group. Model group compared with control group * p < 0.05, ** p < 0.01, *** p < 0.001, HSD-containing serum-treated group compared with the model group $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, Reverse validation group compared with the HSD-containing serum-treated group & p < 0.05, && p < 0.01, &&& p < 0.001.

Article Snippet: The main materials that were used in this study were LPS (L8880, Solarbio), MHY1485 (MHY) (M9050-5MG, AbMole), RIPA buffer (high) (R0010, Solarbio), primary antibodies β-Actin and CD86 (42kDa, BM0627; 70kDa, BM4121, BOSTER), Arg1 and mTOR (40kDa, GB11285-100; 289kDa, GB111839-100, Servicebio), p-mTOR and HIF-1α (289kDa, 5536T; 120kDa, 48085S, Cell Signaling Technology), IL-1β (31kDa, 16806-1-AP, Proteintech), Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies (ZB-2301; ZB-2305, Beijing Zhong Shan-Golden Bridge Biological Technology), Glucose (GLU) Assay Kit, Lactic Acid (LD) assay kit, ATP assay kit, Lactate dehydrogenase (LDH) assay kit (A154-1-1; A019-2-1; A095-1-1; A020-2-2, Nanjing Jiancheng Bioengineering Institute), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (E-BC-K650-M, Elabscience), BCA Protein Assay Kit (PC0020, Solarbio), ELISA Kit for Hexokinase 2 (HK2) activity based on enzyme standard of HK2, ELISA Kit for Tumor Necrosis Factor alpha (TNF-α) (MM-45418M1; MM-0132M1, MEIMIAN), ELISA Kit for Interleukin 6 (IL-6) (E-MSEL-M0001, Elabscience) and ELISA Kit for Pyruvate kinase isozymes M2 (PKM2) (SEA588Mu, Cloud Clone Corp), Cell Staining Buffer, PE Anti-Mouse F4/80 Antibody [CI:A3-1], PE/Cyanine7 Anti-Mouse CD86 Antibody [GL-1], Intracellular Fixation/Permeabilization Buffer Kit and APC Anti-Mouse CD206 Antibody (E-CK-A107; E-AB-F0995D; E-AB-F0994H; E-CK-A109; E-AB-F1135E, Elabscience).

Techniques: Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Biomarker Discovery

Journal: Journal for immunotherapy of cancer

Article Title: XTX101, a tumor-activated, Fc-enhanced anti-CTLA-4 monoclonal antibody, demonstrates tumor-growth inhibition and tumor-selective pharmacodynamics in mouse models of cancer.

doi: 10.1136/jitc-2023-007785

Figure Lengend Snippet: Figure 4 In vitro function of XTX101. (A,B) In vitro inhibition of human CTLA-4 binding to CD80 (A) and CD86 (B) by XTX100 (black), XTX101 (red), and activated XTX101 (blue), as assessed by ELISA. (C) In vitro activity of intact (red) and activated XTX101 (blue) compared with XTX100 (black) in antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay. (D) IL-2 production of SEB-stimulated human peripheral blood mononuclear cells (PBMCs) incubated with XTX100 (black), XTX101 (red), and activated XTX101 (blue), as measured by ELISA. Fold-change from isotype at highest mAb concentration is reported. ND, not determined.

Article Snippet: Serial dilutions of test articles were added to the washed ELISA plates followed by addition of a 2.6 μg/ mL solution of recombinant human CD80 or CD86 (9050- B1- 100 or 9090- B2- 100, R&D Systems).

Techniques: In Vitro, Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Bioassay, Incubation, Concentration Assay

Journal: Journal of biomedical science

Article Title: The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling.

doi: 10.1186/s12929-023-00941-3

Figure Lengend Snippet: Fig. 5 B7-1 and B7-2 dimer interface mimetic peptides attenuate engagement of CD28 by the cognate B7 costimulatory receptor. A In cartoon model of the extracellular domain of costimulatory receptor B7-1 (CD80) (green; 1dr9.pdb), a double beta-barrel, amino acid residues forming pB1-8 are modeled in sticks, with 2 residues making homodimer interface contacts shown in yellow. B, C pB1-8 selectively attenuates intercellular B7-1/CD28 engagement (B) but not B7-2/CD28 engagement (C). Receptor engagement was assayed by flow cytometry as in Fig. 3K for B7-1/CD28 engagement and as in Fig. 3D for B7-2/CD28 engagement. D In the extracellular domain of B7-1, amino acid residues forming pB1-78 are modeled in sticks, with 4 residues making homodimer interface contacts shown in yellow and orange. E, F pB1-78 selectively attenuates intercellular B7-1/ CD28 engagement (E) but not B7-2/CD28 engagement (F). G, H pB2-7 selectively attenuates intercellular B7-2/CD28 engagement (H) but not B7-1/ CD28 engagement (G). Data are mean and SEM of three independent experiments (contour plots: Additional file 1: Fig. S6). Intercellular receptor engagement was compared using the one-tailed unpaired student’s t-test; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001; n.s, not significant

Article Snippet: Recombinant human B7-2 (CD86) Fc chimera and human CD28 Fc chimera expressed in mouse myeloma NS0 cells (R&D Systems) comprise the extracellular 20–239 and 19–152 amino acid domain, respectively, of the mature human ligands fused to C-terminal human IgG1 Fc and are homodimers, disulfide-linked in the Fc domain.

Techniques: Flow Cytometry, One-tailed Test