Journal:

Article Title: Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-terminal immunodominant epitope of the amyloid ? peptide

doi: 10.1016/j.molimm.2010.09.012

Figure Lengend Snippet: Kinetic constants and affinities of various immobilized rFab with Aβ 1–16 -Im7.

Article Snippet: Two different assay formats were designed to analyze the binding of Fab to Aβ: (1) Capture Assay : recombinant Aβ 1–16 -Im7 fusion protein, used as a “surrogate Aβ antigen” (for details, see Section 3), was injected over Fab captured on the surface of a GLC chip containing previously immobilized Human Fab Binder (GE Healthcare); and (2) Direct Assay : recombinant Fabs were injected over various Aβ forms captured on a NeutrAvidin coated chip (NLC, BioRad).

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Downregulated Platelet miR-1233-5p in Patients with Alzheimer’s Pathologic Change with Mild Cognitive Impairment is Associated with Aβ-Induced Platelet Activation via P-Selectin

doi: 10.3390/jcm9061642

Figure Lengend Snippet: Role of let-7i-5p, miR-125a-5p, and miR-1233-5p in the Aβ 1-40 -induced P-selectin expression in platelets and MEG-01 cells. ( a ) Platelet activation status at basal level (gray curve) and after 10 μM Aβ 1-40 stimulation (black curve) was monitored by flow cytometric measurement of PE-P-selectin expression. Data are mean ± SEM of six experiments. (b and c) Overexpression or inhibition of let-7i-5p, miR-125a-5p, and miR-1233-5p expression resulted in the alteration in the level of P-selectin in MEG-01 cells. Let-7i-5p, miR-125a-5p, and miR-1233-5p were individually overexpressed ( b ) or downregulated ( c ) using a specific mimic or inhibitor in MEG-01 cells after treatment with or without 10 μM Aβ 1-40 for 1 h. P-selectin level was modulated in these samples as compared to that in samples treated with MEG-01 negative control (NC). Data are mean ± SEM of six experiments.

Article Snippet: For the adhesion assay, washed platelets or MEG-01 cells were added to a glass bottom dish pre-coated with fibronectin (Sigma-Aldrich) for 1 h. The cells were washed and incubated at 2.5 × 10 5 cells/mL density for 1 h in the presence or absence of Aβ 1-40 and an anti-human P-selectin monoclonal antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Activation Assay, Over Expression, Inhibition, Negative Control

Journal: Journal of Clinical Medicine

Article Title: Downregulated Platelet miR-1233-5p in Patients with Alzheimer’s Pathologic Change with Mild Cognitive Impairment is Associated with Aβ-Induced Platelet Activation via P-Selectin

doi: 10.3390/jcm9061642

Figure Lengend Snippet: Role of P-selectin in the adhesion of platelets or MEG-01 cells. ( a ) Adhesion of platelets (green) was quantified following stimulation with 1, 3, and 10 μM Aβ 1-40 for 1 h with or without 1 μg/mL anti-P-selectin monoclonal antibody pre-treated for 30 min on fibronectin-coated coverslips. Data are mean ± SEM of five experiments. ( b ) Adhesion of MEG-01 cells (top, green fluorescence; bottom, phase-contrast image) was quantified following stimulation with 1, 3, and 10 μM Aβ 1-40 for 1 h with or without 1 μg/mL anti-P-selectin monoclonal antibody pre-treated for 30 min on fibronectin-coated coverslips. (insert) Arrowhead indicates spreading of MEG-01 cells. Data are mean ± SEM of six experiments.

Article Snippet: For the adhesion assay, washed platelets or MEG-01 cells were added to a glass bottom dish pre-coated with fibronectin (Sigma-Aldrich) for 1 h. The cells were washed and incubated at 2.5 × 10 5 cells/mL density for 1 h in the presence or absence of Aβ 1-40 and an anti-human P-selectin monoclonal antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Fluorescence

Journal: British Journal of Pharmacology

Article Title: Protein inhibitor of activated STAT1 Ser 503 phosphorylation‐mediated Elk‐1 SUMOylation promotes neuronal survival in APP/PS1 mice

doi: 10.1111/bph.14656

Figure Lengend Snippet: Elk‐1 is SUMO‐modified by PIAS1 endogenously in the hippocampus. (a) Flag‐vector, Flag‐Elk‐1WT (with or without SUMO1 mutant protein added to the reaction), individual Flag‐Elk‐1 mutant plasmid, or Flag‐Elk‐1 3KR mutant plasmid was transfected to rat hippocampal CA1 area, and Elk‐1 SUMOylation was determined by SUMOylation assay 48 hr later. Anti‐Elk‐1 antibody (left) and anti‐SUMO1 antibody (middle) were used for immunoblotting after immunoprecipitation with anti‐Elk‐1 antibody. Immunoprecipitation and western blot using anti‐Flag antibody confirm the transfection and expression of Flag‐tagged plasmids (middle‐lower panel). Flag‐vector, Flag‐Elk‐1WT, or Flag‐Elk‐1 3KR plasmid was transfected to rat CA1 area, and tissue lysate was immunoprecipitated with anti‐Flag antibody and immunoblotted with anti‐SUMO1 antibody. An additional group of rats received Flag‐Elk‐1WT transfection but was immunoprecipitated with IgG to serve as the control. Tissue lysate was similarly immunoprecipitated and immunoblotted with anti‐Flag antibody to confirm the transfection and expression and Flag‐tagged plasmids (right‐lower panel). (b) The quantified result of Elk‐1 SUMOylation (n = 5 different animals for each group). * P < 0.05 for Elk‐1K229R versus Elk‐1WT group and ** P < 0.05 for Elk‐1 3KR versus Elk‐1WT group and for Elk‐1WT + SUMO1 mutant protein versus Elk‐1WT group. (c) Control siRNA or PIAS1 siRNA was transfected to the rat CA1 area, and endogenous Elk‐1 SUMOylation was determined 48 hr later as described above but without the addition of the recombinant PIAS1 protein. (d) The quantified result of Elk‐1 SUMOylation (n = 5 different animals for each group). # P < 0.05. (e) Western blot of PIAS1 expression and the quantified result (n = 5 different animals for each group). # P < 0.05. (f) Immunohistochemistry showing PIAS1 (green) and Elk‐1 (red) are both present in the nucleus of the same neurons in the CA1 area of the rat brain (n = 3 different animals for each group). The lower panel is the magnification of the selected area from the upper panel. Scale bar is 25 μm for the upper panel and 10 μm for the lower panel. Data are mean ± SEM

Article Snippet: Materials Recombinant human Aβ (1‐42) protein was purchased from GenScript (Piscataway, NJ).

Techniques: Modification, Plasmid Preparation, Mutagenesis, Transfection, Western Blot, Immunoprecipitation, Expressing, Control, Recombinant, Immunohistochemistry

Journal: British Journal of Pharmacology

Article Title: Protein inhibitor of activated STAT1 Ser 503 phosphorylation‐mediated Elk‐1 SUMOylation promotes neuronal survival in APP/PS1 mice

doi: 10.1111/bph.14656

Figure Lengend Snippet: PIAS1 is phosphorylated at Thr487 and Ser503 by MAPK/ERK and acute Aβ induces MAPK/ERK activation and PIAS1 Ser503 phosphorylation. (a) LC–MS/MS chromatography prediction of potential phosphorylation sites on PIAS1 at Thr487 (left) and Ser503 (right). (b) His‐tagged PIAS1WT, PIAS1T487A mutant, and PIAS1S503A mutant recombinant proteins (1 μg each) were incubated with activated GST‐ERK2 protein (30 ng) and 6 μCi of [γ‐32P] ATP (100 μM) for 30 min for kinase reaction and Western blot using anti‐His and anti‐GST antibody. (c) His‐tagged PIAS1 fusion protein (1 μg) was incubated with or without activated GST‐ERK2 (30 ng) and ATP (100 μM) for 30 min for kinase reaction and Western blot. Antibodies for p503PIAS1, PIAS1, and GST were used for Western blot. The p503PIAS1 antibody recognizes phosphorylated PIAS1 only. Results are from two independent experiments for (a), (b), and (c). (d) NH4OH (1%) or Aβ (14 μg) was injected to rat hippocampal CA1 area, and the expression level of pERK1/2, ERK1/2, pSer503 PIAS1, and PIAS1 was determined by Western blot 30 min later. (e) The quantified results of the above measures (n = 6 different animals for each group). ** P < 0.05 for Aβ versus NH4OH group. Data are expressed as mean ± SEM

Article Snippet: Materials Recombinant human Aβ (1‐42) protein was purchased from GenScript (Piscataway, NJ).

Techniques: Activation Assay, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Chromatography, Mutagenesis, Recombinant, Incubation, Western Blot, Injection, Expressing