Anti nkg2d antibody

Anti-ULBP1, anti-ULBP2 and anti-ULBP3 antibodies were purchased from R&D systems (catalog numbers: MAB1380, 1248 and 1517 respectively) and used both for flow cytometry and for ELISA assays. Anti-ULBP1 antibody, (catalog number Sc33564, Santa Cruz Biotechnology) was used for western blotting of ULBP1. The W6/32 mAb was used for MHC-I staining. Anti-NKG2D antibody was purchased from R&D Systems (MAB139). The anti-CD99 (12E7) was used as an isotype control. Anti-CD107a (LAMP-1) was purchased from BioLegend (catalog number 328620). Anti-CD56 (Becton Dickinson) and anti-CD3 (BioLegend) antibodies were used to determine NK purity. Anti-VP2/3 and agnoprotein antibodies were produced in house as well as the rabbit polyclonal antibodies against VP1. Anti-T-Ag antibody was purchased from Abcam (Pab416). The commercial recombinant ULBP-1 Fc chimeric protein (R&D systems, catalog number 1380-UL) was used for the generation of ULBP1 standard curve.
CD16-Ig, NKp30-Ig and NKp46-Ig and NKG2D-Ig fusion proteins were generated in the human embryonic kidney 293T cells and were purified on a protein G column as described [38 (link)]. The fusion proteins used in this work were regularly assayed by SDS-PAGE protein gels, to ensure that the proteins were not degraded. Protein purity of all Ig fusion proteins used in this study was approximately 100%.

This blocking assay was based on methods previously reported (15 (link)). HES-fractionated BM cells from HDs and patients with FA were divided into 2 equal aliquots (range: 0.5 × 10⁶ to 1 × 10⁶ cells) and resuspended in 200 μL IMDM supplemented with 20% FBS and loaded into V-bottomed 96 MicroWell plates (Nunc). One of the aliquots was incubated with mouse IgG1 (25 μg/mL) as an isotype control (clone 11711), and the other one was incubated with the anti-NKG2D antibody (25 μg/mL) to block the NKG2D-activating receptor (clone 149810), both from R&D Systems. Cells were incubated for 10 minutes at RT and resuspended after 5 minutes. Subsequently, cells were gently centrifuged at 60g for 3 minutes to facilitate cell interaction at the well bottom and were then further incubated at 37°C in 5% CO₂ for 4 hours. Cells were then collected, resuspended, and seeded in methylcellulose cultures (see experimental scheme in Figure 6). Colony numbers generated in the NKG2D-blocking group were normalized with respect to numbers corresponding to the isotype control group. Some experiments were conducted using purified CD56⁺ cells (effectors cells) and purified CD34⁺ cells (target cells) sorted by FACS (see above). In these experiments, a ratio of 10:1 (effector/target cells) was used.