NKL cells (5×10
6 cells/condition) electroporated with CD3ζ siRNA or control siRNA were stimulated with 5μg/ml of anti-CD16 (3G8) and 2.5μg/ml of
goat anti-mouse crosslinker (EMD Millipore) or 5 ×10
4 K562 cells for one minute. Stimulation was stopped by washing cells twice with cold PBS. In the case of murine NK cells, 3×10
6 IL-2 stimulated NK cells were stimulated using 3×10
4 YAC-1 cells. Total protein was extracted using RIPA buffer plus proteinase and phosphatase inhibitors. For Western blot anti-p-Tyr (PY20, Santa Cruz Biotechnology),
anti-CD3ζ (6B10.2, Santa Cruz Biotechnology) and
anti-β actin (Abcam) were used. Images were acquired with a FUJI LAS-4000 imager (GE Healthcare Life Sciences) and analyzed using FIJI. Lanes from Western blot of lysates from murine NK cells were cut and pasted to place together with their control.
Suárez-Fueyo A., Bradley S.J., Katsuyama T., Solomon S., Katsuyama E., Kyttaris V.C., Moulton V.R, & Tsokos G.C. (2018). Downregulation of CD3ζ in Natural Killer Cells from Systemic Lupus Erythematosus Patients Confers a Pro-Inflammatory Phenotype. Journal of immunology (Baltimore, Md. : 1950), 200(9), 3077-3086.
