Journal: Molecular Medicine Reports

Article Title: The effect of anti-HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

doi: 10.3892/mmr.2021.11994

Figure Lengend Snippet: Effect of anti-HLAI on ICAM-1, HLA-DR, CD46 and CD59, and the impact of halofuginone or everolimus treatment. (A) Representative experiment for each of the evaluated factors. (B) Cumulative results are presented. Anti-HLAI antibodies upregulated ICAM-1, HLA-DR, CD46 and CD59. Halofuginone or everolimus treatment decreased ICAM-1. Data are presented as the mean ± SEM. *P<0.05 vs. control cells, # P<0.05 vs. anti-HLAI-treated cells, ^ P<0.05 vs. anti-HLAI-treated cells administered halofuginone, + P<0.05 vs. anti-HLAI-treated cells administered everolimus and $ P<0.05 vs. anti-HLAI-treated cells with halofuginone and everolimus. HLAI, human leukocyte antigen class I; ICAM-1, intracellular adhesion molecule-1; Hal, halofuginone; Ever, everolimus; Ctrl, control.

Article Snippet: Blots were incubated at 4°C for 16 h with the primary antibodies specific against activated cleaved caspase-3 (cleaved caspase-3, 1:1,000, cat. no ab13847, Abcam), focal adhesion kinase (FAK, 1:100, cat. no sc-271126, Santa Cruz Biotechnology, Inc.), phosphorylated at Tyr397 FAK (p-FAK, 1:1,000, cat. no 8556, Cell Signaling Technology, Inc.), mTOR (1:100, cat. no sc-517464, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser2448 mTOR (p-mTOR, 1:100, cat. no sc-293133, Santa Cruz Biotechnology, Inc.), p70S6 kinase (p70S6K, 1:100, cat. no sc-8418, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr389 p70S6K (p-p70S6K, 1:1,000, cat. no 9234, Cell Signaling Technology), protein kinase B (Akt, 1:100, cat. no sc-5298, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser474 Akt (p-Akt, 1:1,000, cat. no 4060, Cell Signaling Technology, Inc.), GCN2 kinase (GCN2K, 1:100, cat. no sc-374609, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K, 1:1,000, cat. no ab75836; Abcam), eIF2α (1:100, cat. no sc-133132, Cell Signaling Technology, Inc.), phosphorylated at Ser51 eIF2α (p-eIF2a, 1:1,000, cat. no 9721, Cell Signaling Technology, Inc.), intercellular adhesion molecule 1 (ICAM-1, 1:1,000, cat. no 4915; Cell Signaling Technology), HLA-DR (Ultra-LEAFTM Purified anti-human HLA-DR Antibody, cat. no 307648, Biolegend), CD46 (1:1,000, cat. no CSB-PA923298, Cusabio), CD59 (1:1,000, cat. no CSB-PA004947YA01HU, Cusabio), and β-actin (1:5,000, cat no. 4967, Cell Signaling Technology, Inc.).

Techniques: Control

Journal:

Article Title: Regulated complement deposition on the surface of human endothelial cells: Effect of tobacco smoke and shear stress

doi: 10.1016/j.thromres.2007.11.005

Figure Lengend Snippet: Changes in cell surface expression of complement regulators (Mean ± S.D.), CR1 (CD35) (n = 3), DAF (CD55) (n = 4), and Protectin (CD59) (n = 4) in response to BMEC exposure to TS (0.03 puffs/ml), chemical (LPS at 1 μg/ml or INF-γ at 100 U/ml), or mechanical shear stress (18 dyne/cm2). Results were normalized to baseline CD35, CD59 and Cd59 expression observed on untreated BMEC monolayers. The data were analyzed by single factor ANOVA. (*) Denotes a statistically significant increase (P<0.05).

Article Snippet: Murine monoclonal anti-human CD35, CD55, and CD59 antibodies were obtained from Ancell Corporation (Bayport, MN), and used at concentrations of 1 μg/ml, 10 μg/ml, and 5 μg/ml, respectively.

Techniques: Expressing

Journal: Journal of Oral Microbiology

Article Title: Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76

doi: 10.1080/20002297.2022.2105013

Figure Lengend Snippet: Mode of receptor recognition of DLY and other CDCs. Each recombinant CDC (rDLY, rSm-hPAF, rILY, and rSLY) was incubated with human erythrocytes in the presence or absence of cholesterol and/or human CD59 monoclonal antibody (YTH53.1). Triplicate samples were assayed at least twice each. Representative results are shown as hemolysis averages with standard deviations (SD). Significance of differences between hemolysis in the absence (dark-gray bar) and presence of inhibitor(s) for receptor binding of CDCs (cholesterol only, cyan bar), YTH53.1 only (magenta bar), and both (purple bar) was evaluated using F-tests followed by Welch’s t-tests or Student t-tests (**p < 0.01, *p < 0.05).

Article Snippet: To inhibit the binding between recombinant CDC and human CD59 on erythrocytes, we pre-incubated anti-human CD59 monoclonal antibody (YTH53.1; DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan) with human erythrocytes at a final concentration of 50 μg/mL for 15 min at 37°C and then washed them with PBS.

Techniques: Recombinant, Incubation, Binding Assay