FIGURE 2.

MS identification of 37βic and siRNA silencing. A, gel section with a silver-stained band (arrow) corresponding to 37βic from IL-2Rβ. Proteins from whole cell lysates of Lαβγ cells were immunoprecipitated using an anti-IL-2Rβ Ab or control Ab and resolved in polyacrylamide gel. After fixation and silver staining as described under “Experimental Procedures,” the excised protein was analyzed by MS. The three bands at 75 kDa correspond to full IL-2R. B, amino acid sequence of transmembrane and C-terminal domains of the IL-2Rβ. The underlined sequences are those identified by MS of protein purified from the excised 37-kDa band. Amino acids of the transmembrane domain are boxed. C, siRNA silencing of 37βic produced in NKL cells. 1 × 10⁷ NKL cells were transfected with the indicated concentrations of siRNA against IL-2Rβ or control siRNA using the Amaxa system. 48 h after transfection, immunoprecipitates were performed using anti-IL-2Rβ Ab and analyzed by Western blot. D, siRNA silencing of cell surface IL-2Rβ in NKL. Cells were transfected with the indicated concentrations of siRNA against the IL-2Rβ or control siRNA. 48 h after transfection, cell surface IL-2Rβ was analyzed by flow cytometry using monoclonal Ab 561. The percentage of cells expressing IL-2Rβ on their surface is presented. In A the arrow shows 37βic, and the line shows full-length IL-2Rβ.