DAP10 stability, glycosylation and association with NKG2D in human NK cells. (A) Primary NK cells were cultured with IL-2 for 3 days, followed by treatment with the translation inhibitor, cycloheximide (CHX) or DMSO (vehicle) for the indicated times. DAP10 protein levels from the total cell lysates were visualized by immunoblotting and protein loading was verified by blotting with anti-actin antibody. (B) NKG2D was immunoprecipitated from NKL cell lysates. The immunoprecipitated proteins were subsequently immunoblotted with anti-NKG2D and anti-DAP10 antibodies as indicated. Isotype specific IgG (cIgG) immunoprecipitation was used as a negative control. (C) NKL cells were treated with an O-glycosylation inhibitor (benzyl-α-GalNAC) for either 1 or 2 days. The total cell lysates were resolved on a 10%-20% gradient gel, followed by anti-DAP10 immunoblotting. (D) NKL cells were treated with benzyl-α-GalNAC for 2 days and NKG2D was immunoprecipitated from the total cell lysates. The immunoprecipitated proteins were immunoblotted with anti-DAP10 and anti-NKG2D antibodies. Isotype specific IgG (cIgG) immunoprecipitation was used as a control.