LFA-1 ligation induced Akt phosphorylation, p47 phox phosphorylation, and Rac-1 activation via a PI3K-dependent pathway. (A) U937 cells (1 × 106 cells for each condition) were preincubated with or without LY294002 (20 μM) and stimulated with TS1-18 and TS1-22 for the indicated time periods. The cell lysates were subjected to Western blot analysis using phospho-Akt antibody or Akt antibody to determine the activated and the total amount of the Akt protein. (B) U937 cells were pretreated with or without the PI3K inhibitor LY294002 (20 μM) for 15 minutes and stimulated with TS1-18 for 1 hour. The cell lysates were immunoprecipitated with rabbit anti-p47phox polyclonal antibody. The precipitants were then resolved with SDS-PAGE and subjected to Western blot analysis using antiphospho-serine antibody or a mouse anti-p47phox mAb. (C) After TS1-18 stimulation, cells were lysed and the Rac-1-activated fraction (Rac-1-GTP) was precipitated with GST-PAK beads. Aliquots of lysates were analyzed along with the precipitates by Western blotting using an anti-Rac-1 mAb. These experiments were repeated 3 times with similar results.
