Identification of proteins associated with 2B4 ITSM peptides. (A) A control peptide (c) or peptides corresponding to the 2B4 ITSMs 1 to 4 either nonphosphorylated (Y1-4) or phosphorylated at the central tyrosine (pY1-4) were coupled to beads and incubated with lysate from primary human NK cells. Associated proteins were separated by 10% to 20% SDS-PAGE and visualized by silver staining. The lower part of the gel was developed longer for greater sensitivity. Protein bands that differed between the peptides were cut out and identified by mass spectrometry-based sequencing. The identified proteins were SHIP (1), SHP-2 (2), SHP-1 (3), Csk (4), SAP (5), and Hsp-70 (7). No sequence could be obtained for band no. 6. The experiment was repeated 3 times with the same results. (B-C) A portion of the samples described in panel A, including the lysate of primary human NK cells, was analyzed by 10% to 20% SDS-PAGE followed by Western blotting using the indicated antibodies.