human apoe e2 e3 target replacement  (Taconic Biosciences)

Journal: The Journal of Biological Chemistry

Article Title: Glycolaldehyde is an endogenous source of lysine N -pyrrolation

doi: 10.1074/jbc.RA120.013179

Figure Lengend Snippet: GA-modified proteins act as a ligand of apoE. A, pulldown assay for detection of binding proteins for GA-modified proteins in mouse serum. BSA coupled to Dynabeads was incubated with 25 mm GA in PBS for 1 or 7 days to obtain GA-modified protein-coupled beads. The mouse serum was incubated with the ligand-coupled beads for 1 h at room temperature. The ligands used were BSA and GA-BSA. Proteins bound to the beads were eluted by addition of sample buffer, heating (95 °C, 5 min), and separation by SDS-PAGE. The proteins indicated by the arrowheads represent thrombospondin 1 (band 1), complement factor H–related protein C (band 2), coagulation factor XIII A chain (band 3), serotransferrin (band 4), antithrombin (band 5), apolipoprotein E (band 6), and metalloproteinase inhibitor 3 (band 7), as identified by LC-MS/MS analysis. B, binding of modified proteins to apoE. Serum proteins bound to the beads were prepared and separated by SDS-PAGE as described in A. ApoE was detected by immunoblotting with anti-apoE mAb E6D7 (Abcam). WB, Western blot. C, binding of GA-modified proteins or pentylamines to the apoE isoforms. The apoE isoforms (2 μg/ml) were immobilized on a plate and incubated with biotin-labeled BSA or GA-BSA (50 μg/ml) (left panel) or with biotin-labeled N-pentylamine (PA) or GA-PA (0.5 mm) (right panel) at 37 °C for 1 h. Data are from single experiments performed in triplicate wells and are representative of three individual experiments. The results shown are means ± S.D. (n = 3). D, pulldown assay for binding of GA-modified proteins to the apoE isoforms. Three apoE isoforms were incubated separately with ligand-coupled beads for 1 h at room temperature. The ligands used were BSA, BDA-BSA, and GA-BSA. Proteins bound to the beads were eluted by addition of sample buffer, heating, and separation by SDS-PAGE. The apoE isoforms were detected by immunoblotting with anti-apoE mAb E6D7. E, levels of the antibody titers against antigens (BSA, BDA-BSA, and GA-BSA) in sera from 12 weeks of male control and spontaneously hyperlipidemic mice. Elevations of IgG (left panel) or IgM (right panel) immune responses in the serum samples were measured by ELISA using native BSA, BDA-BSA, and GA-BSA as the coating antigens. *, p < 0.05; **, p < 0.01. The results shown are means ± S.D. (n = 3).

Article Snippet: The recombinant human apoE isoforms (E2, E3, and E4) were obtained from PeproTech.

Techniques: Modification, Binding Assay, Incubation, SDS Page, Coagulation, Liquid Chromatography with Mass Spectroscopy, Western Blot, Labeling, Control, Enzyme-linked Immunosorbent Assay