Figure 3.
Impact of inhibitors on components of the phox complex. (A) Translocation of p47phox to membranes. Neutrophils were incubated with DMSO (D) or 2 at 37°C for 30 min and stimulated with TNF (T), PMA (P), or buffer (NS). After 40 min, cells were lysed and membrane fractions collected by ultracentrifugation, separated by SDS-PAGE, and Western-blotted with anti-p47phox antibody. (B) Activation of Rap1A. Lysates of neutrophils that had been pretreated for 30 min with DMSO (D), 2, KH7 (25 μM), or the tmAC inhibitor ddAdo (25 μM), and then stimulated for 30 min with TNF or buffer (NS) alone were incubated with agarose beads coupled to recombinant RalGDS-Rap binding domain to affinity purify GTP-bound Rap1A. Beads were boiled in SDS sample buffer and the supernate subjected to SDS-PAGE and Western blot with anti-Rap1A antibody (top row). Western blot of total cell lysates (bottom row) served as a control.