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Pipeline publication

[…] face. Then, cells were treated with 0.3% Triton X-100, and re-incubated with anti-FLAG M2 mAb, followed by Alexa448-labeled anti-mouse IgG (Invitrogen), to detect intracellular KIR2DL5 molecules. After this staining strategy, based on Ref. (), cells were visualized on poly-l-lysine-coated glass-bottom dishes on a confocal laser-scanning microscope (TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany) using Argon (488 nm) and Helium-Neon (543 nm) lasers and a 20×/0.5 lens. Images were acquired using the LAS AF SP5 software (Leica)., We based on the KIR2DL1 X-ray structure () (PDB accession code: 1NKR) to predict the structures of the D2 Ig-like domains of KIR2DL5A*001 and *005, using the SWISS-MODEL automated protein structure homology-modeling system (), accessible via the ExPASy web server. Quality of each model was assessed with the QMEAN Z-score (), which describes the likelihood that a model quality is comparable to high-resolution experimental structures. Structures were visualized and edited using the PyMOL Molecular Graphics System (version 1.7.4, Schrödinger, LLC)., Flow cytometry assays of PBMCs from donors with a KIR2DL5A*001 allele identify discrete subpopulations of NK cells reacting with the only available anti-KIR2DL5 mAb, UP-R1 (average of positive NK cells in 11 donors ± SD: 5.18 ± 2.15%; range: 1.71–7.89%), consistently with the clonal expression pattern reported for the receptor (, ). In contrast, much fewer UP-R1-positive NK cells are seen in donors carrying allele KIR2DL5A*005 (0.50 ± 0.24%; range: 0.23–0.93%; n = 9; p = 0.0001), their proportions being not significantly different from the background seen in donors lacking a transcribed KIR2DL5 gene (0.33 ± 0.20%; range: 0.1 […]

Pipeline specifications

Software tools SWISS-MODEL, QMEAN, PyMOL
Organisms Homo sapiens
Chemicals Amino Acids, Amino Acids, Dicarboxylic, Excitatory Amino Acids, Amino Acids, Acidic, Glycine