Paxillin coprecipitation with α4 integrins correlates with the expression of high-affinity α4 integrins. (A) Flow cytometry profiles reveal slightly higher cell surface expression of α4 integrins on unstimulated Jurkat compared with U937 cells (top row), whereas VCAM-1/Fc binding to Jurkat cells is much higher (bottom row). (B) Western blotting for paxillin (top panel), talin (middle panel), and α4 integrins (bottom panel) of Jurkat and U937 cell lysates (1 mg) immunoprecipitated with HP2/1 antibody to α4 integrins (left blots) demonstrates coprecipitation of paxillin and talin with Jurkat, but not U937 α4 integrins. Western blotting of total cell lysates (20 μg/lane, right blots) illustrates abundant expression of all 3 proteins in both cell types. Representative data from 1 of 5 experiments are shown. (C) Coprecipitation of paxillin with α4 integrins was detected in 1, 0.3, and 0.1 mg Jurkat cell lysate, but not in the U937 cell lysate, even though the quantity of α4 integrins precipitated from U937 cells was comparable, or even exceeded that in the 0.3- and 0.1-mg Jurkat cell lysates. Representative data from 1 of 4 blots are shown.