PILAR is a ligand for CD161. (A) 293 cells transfected with HA-PILAR and K562 cells transduced with CD161 are identified as “+,” whereas “−” denotes the parental cell line. Cell extracts were incubated for 2 hours and immunoprecipitation (IP) was performed with either anti-HA Ab (HA11; left; Covance, Berkeley, CA) or anti-CD161 Ab (right), plus protein G/protein A agarose beads. The immunoprecipitates were run in a Western blot and analyzed for their reactivity against the opposite Ab. A positive control including either CD161⁺ cells (left) or PILAR⁺ cells (right) without IP was also included. (B) Coincubated PILAR⁺ and CD161⁺ cell extracts immunoprecipitated with an irrelevant Ab (iAb; left), or coincubated lysates from K562 cells ectopically expressing CD161 plus 293 cells expressing an irrelevant HA-tagged sequence (right) did not contain CD161. A positive control including CD161⁺ cells was included. (C) Immunoprecipitates of coincubated lysates from CLEC2D⁺ and CD161⁺ cells performed with an anti-CD161 Ab contained the expected approximately 25-kDa CLEC2D band. A positive control, including CLEC2D⁺ cells (left) without IP, was included. (D) Pilar transduced/untransduced (+/−) K562 cells were incubated with a CD161-Myc (left) or a control-Myc (GenBank no. NM_007048; right) protein (0.5 μg/mL, 45 minutes). Specific binding was confirmed 3 times with an anti–Myc-FITC Ab (Sigma-Aldrich, St Louis, MO).