Association of α4 with PP2Ac. (A) Cell lysates of Jurkat were immunoprecipitated for 2 hours at 4°C with either anti-hα4 Ab, anti-PP2Ac Ab, or preimmune serum. The immunoprecipitates were separated by SDS/10% PAGE and transferred to a nitrocellulose filter. The filter was then probed with anti-PP2Ac Ab. The migration of human PP2Ac is indicated. (B) Cell lysates of Jurkat (50 × 106 cells) were mixed with 10 μg of GST-PP2Ac fusion protein or the control GST alone. The precipitates (15 × 106 cell equivalents per lane) were captured by Glutathione-Sepharose beads, separated by SDS/10% PAGE, and transferred to nitrocellulose filter. The filter was immunodetected with anti-hα4 Ab. Jurkat cell lysate (1 × 106/lane) was used as a positive control for α4 immunoblotting. The migration of α4 is indicated. (C) mα4 was synthesized in vitro in the presence of 35S-methionine using an in vitro translation kit (Amersham). GST-PP2Ac or GST alone was mixed with radiolabeled α4 and precipitated by Glutathione-Sepharose. Recovered proteins were separated by SDS-PAGE and subsequently developed by autoradiography. (D) Schematic diagram of GST-α4 fusion proteins used to localize the region that is necessary for binding to PP2Ac. Numbers on the left side indicate the positions of amino acid residues of α4. Restriction enzyme sites used for construction of mutants are shown. Ba,BamHI; EI, EcoRI; EV, EcoRV; HII,HincII. Hatched regions indicate the binding site. The intensities of the PP2Ac bands in the pull-down assay performed in (E) are indicated by ++, +, and −. (E) Various mutants of GST-α4 fusion proteins were tested for their binding activities to PP2Ac.
