Human erythrocyte membranes were solubilized using 1% Nonidet P-40 and subjected to gel-filtration on Sephacryl S-300, and fractions no. 40 through 51 were then used for immunoprecipitation with antiphosphotyrosine (PT66). Aliquots (90 μL) of the immunoprecipitated, immobilized proteins were incubated in protein kinase buffer containing 1.0 μmol/L [γ-³²P]ATP (3.7 × 10⁷ dpm) and 20.0 mmol/L magnesium acetate or 10.0 mmol/L manganese acetate, in the absence or presence of 1 nmol/L IP₄ and/or anti-FKBP12 (1 to 1,500), as indicated. After stopping the reactions, proteins were applied to 3% to 17% gradient SDS polyacrylamide gels. Labeling was quantitated by the densitometric scanning (Molecular Dynamics Model 300B computing densitometer) of autoradiograms. Data are reported in arbitrary units (sum above background). Labeling of the 30-, 36-, 42-, 60-, 72-, and 165-kD proteins was virtually unaffected by these ligands, and data for the 36- and 42-kD proteins are shown. Aliquots of the suspension of agarose-linked antiphosphotyrosine (PT66) that had not been exposed to the protein preparation were carried through these procedures to assess background labeling and/or nonspecific binding. These studies were repeated 4 times with different membrane preparations, and the data obtained were quantitatively the same.