High-affinity but not low-affinity α4 integrins in Jurkat cells are dephosphorylated on Ser988 and are associated with paxillin. A representative experiment (1 of 4) of sequentially probed Western blots is shown. In lane 1, both high- and low-affinity α4 integrins in 1 mg Jurkat cell lysate were precipitated by HP2/1 antibody. Phosphorylation of Ser988 and association of paxillin and talin with α4 is seen. Selective precipitation of high-affinity α4 integrins with VCAM-1/Fc in lane 2 (4 mg lysate) reveals that these integrins are not phosphorylated but are associated with paxillin and talin. In contrast, in lane 3 low-affinity integrins precipitated by HP2/1 from 1 mg lysate depleted of high-affinity integrins were phosphorylated on Ser988 and were not associated with paxillin. Lane 4 shows 20 μg total Jurkat cell lysate.