Figure 2.
Function of HS1 in cytolysis. (a) HS1 knockdown in NKL cells and primary NK cells expressing a tetracycline-inducible shRNA sequence targeting HS1 (HS1A or HS1 B) or a nontargeting control shRNA (HS1sc) and treated for 0–72 h (above lanes) with doxycycline; HS1 was immunoprecipitated and analyzed by immunoblot with anti-HS1. (b) Restoration of HS1 expression in HS1-knockdown cells treated with doxycycline and then transfected by nucleofection 72 h later with shRNA-resistant GFP-tagged wild-type or mutant HS1 constructs (above lanes), followed by immunoprecipitation of HS1 at 72 h later. For immunoblot analysis of phosphorylated tyrosine, cells were treated with 2 mM Na3VO4 for 15 min before lysis. HS1 was immunoprecipitated and analyzed by immunoblot with anti-HS1. WCL (a,b), immunoblot of one tenth of the preimmunoprecipitation whole-cell lysate with anti-actin to show equal loading of lanes, (c) Cytolysis assays of NK cell populations and K562 target cells combined for 4 h at 37 1C, presented as the killing index, based on the release of adenylate kinase into the media. WT, wild-type; KD, knockdown. *, P < 0.01. Data are representative of at least three separate experiments (mean and s.e.m. of triplicate wells, c).