A specific mAb directed against the membrane distal domain (D1) of NKp46 does not block NK-mediated lysis of target cells. (A) NK cells (NK line), BW, and BW transfected with NKp46 (BW/NKp46) were incubated with or without 461-G1 mAb (thick black line) or control 12E7 mAb (gray histogram). Shown is one representative experiment of 3 performed. (B) ELISA plates were coated with 2 μg/mL of fusion proteins NKp30-Ig, NKp46D2-Ig, NKp46D1-Ig, NKp46-Ig, KIR2DL1-Ig (KIR-I-Ig), and KIR2DL2-Ig (KIR-II-Ig). Fusion proteins were incubated with no antibody or with 461-G1 for 1 hour on ice. HRP-conjugated rabbit antimouse IgG diluted 1:2500 was used as secondary mAb. PBS was used as negative control. Shown is one representative experiment of 3 performed. (C) The indicated proteins (10 μg), NKp30-Ig (1), NKp46D2-Ig (2), NKp46D1-Ig (4), NKp46-Ig (5), KIR2DL1-Ig (6), and the control low-protein medium (LPM [3]) were analyzed on SDS-PAGE. The marker is indicated by M. Fusion proteins were analyzed with the 461-G1 mAb and HRP-conjugated goat antimouse Ig antibodies. Shown is one representative experiment of 2 performed. (D) ³⁵S-labeled P815 cells were incubated either with no mAb (□), anti-CD16 mAb (B73.1.1; ♦), anti-NKp46 mAb (461-G1; ○), or anti-CD99 mAb (12E7; ▴) for 1 hour on ice. NK cells were next added in effector to target ratio (E/T) of 3:1. Shown is one representative experiment of 7 performed. (E) NK cells were preincubated with the indicated mAb for 1 hour on ice, washed and incubated with either uninfected 1106mel or 1106mel infected with various influenzas as indicated. This experiment was preformed in E/T ratio of 20:1 and represents one of 4 performed.