NKp44L immune-detection. (A) Direct western blot in the presence of nonreduced or reduced lysates. Proteins from Jurkat cells were extracted with radioimmunoprecipitation assay (RIPA) or NET buffer, treated or untreated with DTT, resolved on an 8% SDS-PAGE and then immunoblotted with anti-NKp44L mAb (αNKp44L). (B-C) Cell lysates from WM1361 cells and PBMCs were immunoprecipitated with anti-NKp44L mAb or the NKp44-Ig fusion protein, and then either (B) immunoblotted with the NKp44-Ig fusion protein or (C) anti-NKp44L mAb (αNKp44L). (B-C) Immunoprecipitations with isotype control are shown in the left panels. (D) WM1361 cell lysates were immunoprecipitated with the anti-N-ter polyclonal MLL5 Ab and immunoblotted with anti-NKp44L mAb (αNKp44L) or the NKp44-Ig fusion protein. C1 and C2 are controls obtained without lysate and without anti-MLL5 Ab, respectively. (E) Cell extracts from biotinylated WM1361 cells or PBMC were immunoprecipitated with anti-NKp44L mAb (αNKp44L) (left panel) or the NKp44-Ig fusion protein (right panel), and then immunoblotted with horseradish peroxidase-streptavidin. (F) Control Immunoprecipitation (IP) after cell-surface biotinylation of WM1361 cells. Cell extracts were immunoprecipitated with anti-actin mAb (α−actin) and then either immunoblotted with horseradish peroxidase-streptavidin (lower panel) or anti-actin mAb (α−actin) (higher panel). Nonimmunoprecipitated whole extract (WE) was immunoblotted with anti-actin mAb (α−actin). (C) Control obtained without lysate. IP, immunoprecipitation; WB, western blot. An arrow indicates the specific band.