Figure 1. SAP partners are exclusively in the membrane fractions.
Jurkat cells were either untreated or treated with 25 µM of pervanadate for 30 minutes. Cells were fractionated by Dounce homogenization and membranes were purified by ultra centrifugation, resolubilized in lysis buffer, precleared and GST or GST-SAP were incubated with the cytosol or membrane fractions. A, pull downs were separated and immunoblotted with anti-phosphotyrosine antibody (4G10). B, crude membrane or cytosolic fractions from 5×105 cells were separated and immunoblotted for SAP. Each band was numerically quantitated (indicated in arbitrary units, a.u.) and the percentages indicate the evolution of the band intensity in the stimulated lysate reported to the non-stimulated lysate in each fraction. These experiments were repeated at least four times. TME: Total Membrane Extract, TCE: Total Cytosolic Extract.