(A) Analysis of JAK3 expression. Lysates of BCLs obtained from normal control subject (lane 1) or JAK3-deficient patients cells (lanes 2 through 5) were subjected to SDS-PAGE and then immunoblotted (IB) with JAK3 antiserum (a-JAK3, top). The membrane was then stripped and reblotted with JAK1 antiserum (a-JAK1, bottom) to verify equal loading. (B) Analysis of JAK3 phosphorylation. Control (lanes 1 through 3 and 10 through 12) and JAK3-SCID patients cells (lanes 4 through 9 and 13 through 15) were stimulated with the indicated cytokine for 15 minutes, lysed, and immunoprecipitated (IP) with a-JAK3. Complexes were detected by immunoblotting (IB) with monoclonal antibody specific for phosphorylated tyrosine residues (a-PY, top). The membrane was then stripped and the presence of JAK3 in the immunoprecipitates was verified by reblotting with a-JAK3 (bottom).