Anti nkg2d

Purified human CD3^-CD56⁺ dNK cells from uninfected, infected, and Tim-3-neutralized infected groups were air-dried onto Poly-L-lysine coated slides. After fixation in 4% paraformaldehyde for 30 min, slides were then blocked with goat serum for 1 h at room temperature. dNK cells were incubated overnight at 4°C with anti-Tim-3 (1/200, Abcam), anti-NKG2D (1/200, Abcam), anti-GranzymeA, anti-GranzymeB and anti-Perforin (1/200, all from Proteintech) or for 45 min with anti-CD56 (clone HCD56, Biolegend) and anti-KIR2DL4 (clone mAb33, Biolegend) antibody. After being washed three times with PBS, cells were then incubated with appropriate concentrations of secondary antibodies for 1 h at 37°C. Cy3 rabbit anti-goat IgM (1/500, Bioss) was used as the secondary antibody for anti-Tim-3 antibody, and Cy3 donkey anti-rabbit IgG (1/500, Bioss) was used as secondary antibody for anti-NKG2D, anti-GranzymeA, anti-GranzymeB and anti-Perforin. Subsequently, dNK cells were stained with the DAPI (nucleic acid stain 4’,6-diamidino-2-phenylindole) for 15 min and washed 3 times with PBS. Finally, confocal microscopy of cells was performed using Zeiss LSM880.

The cells were washed in PBS and immediately lysed using cell lysis buffer containing 1% Triton X-100, 1% NP40, 50 mM Tris-Cl pH 8, 150 mM NaCl, 2 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, phosphatase inhibitor cocktail (Sigma), and complete protease inhibitor cocktail (Roche). Protein concentration was quantified by BCA assay (Bio-Rad). Equal amounts of protein were resolved on an SDS-PAGE gel and then transferred onto nitrocellulose membranes (Millipore, Bedford, USA). After blocking in 5% skimmed milk for 30 min, the membranes were incubated with primary antibodies, such as anti-MAGT1 (Abcam, 1:1000), anti-PD-1 (Abcam, 1:1000), anti-NKG2D (Abcam, 1:1000), anti-β-actin (Abcam, 1:1000), and anti-GAPDH (CST, 1:1000), at 4 °C overnight. After washing, the membrane was incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, USA; 1:1000) at RT for 1 h. The signal was detected using enhanced chemiluminescence system (Millipore). The band intensity was analyzed with Image J software (NIH, USA) and the protein levels were normalized by GAPDH or β-actin.