(A) Immunoblot analysis of PLCγ1 in membranes and cytosolic fractions from resting T lymphocytes. Proteins from membrane and cytosolic fractions were separated by SDS-PAGE, blotted onto nitrocellulose, and reacted with anti-PLCγ1 antibody. The ECL system revealed for each fraction one band apparently at the same molecular weight. Control is represented by a cell lysate from a PLCγ1⁺ line (Jurkat); 145 corresponds to molecular weight (in kilodaltons) assessed on the basis of standard comigration. (B) Tyrosine phosphorylation level of PLCγ1 immunoprecipitated from membranes and cytosolic fractions. PLCγ1 immunoprecipitated from membrane and cytosolic fractions was probed with anti-phosphotyrosine antibody (PY-20) and revealed by the ECL system. Membrane PLCγ1 was consistently phosphorylated, while no or weak reactions were detected in the cytosolic enzyme. After stripping anti-PY-20, the same blot was reprobed with anti-PLCγ1 antibody to show that the levels of the protein loaded on the gel were comparable in both samples. Result is representative of 3 different experiments.