Non fat dried milk

Equal amounts of protein samples were separated on SDS-PAGE gels (7.5–10%) and electro-transferred onto polyvinylidene difluoride membranes (BioRad Laboratories, Hercules, CA, USA) which blocked with 5% non-fat dried milk (Santa Cruz Biotechnology, Dallas, TX, USA) or bovine serum albumin (Sigma Aldrich, Saint Louis, MO, USA) in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for 1 h at room temperature on a rotator. Then, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with alkaline phosphatase-linked goat anti-rabbit or anti-mouse antibodies for 1h at RT. The list of primary and secondary antibodies is enclosed as Supplementary Materials (Tables S3 and S4). The bands were visualized using 1-Step™ NBT/BCIP Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) and their intensities were semi-quantitatively calculated with ImageJ software (https://imagej.nih.gov/ij/, National Institutes of Health, Bethesda, MD, USA). All experiments were repeated thrice.

To confirm whether artemisinin has an effect on the downstream signaling of activating receptors in NK cells, the levels of phosphorylated and total Vav-1 and ERK 1/2 proteins were probed. NK-92MI cells were treated with 0.1 μM artemisinin for 1, 5, 10, or 15 min. After collecting cells, cells were lysed in lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing PMSF and phosphatase inhibitor cocktail on ice. Proteins were equally loaded on SDS-PAGE, and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). Blocking solution containing 5% non-fat dried milk (Santa Cruz Biotechnology, Delaware, CA, USA) was used to block the membrane for 30 min. The membrane was incubated with rabbit polyclonal anti-human antibody to detect total Vav-1 and phosphorylated Vav-1 (Santa Cruz Biotechnology, Delaware, CA, USA), or with rabbit anti-human antibody to detect total ERK 1/2 and phosphorylated ERK 1/2 (Cell Signaling Technology) overnight at 4 °C. After washing with PBS containing 0.1% Tween-20, the membrane was stained with goat anti-rabbit IgG peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Proteins were detected using Amersham ECL Western blotting detection reagent (GE Healthcare, Little Chalfont, UK). The signal was detected using a chemiluminescence-imaging device LAS-3000 (Fujifilm, Tokyo, Japan).