FIGURE 3.

Rap1 activation by FcγR ligation is independent of both cAMP and Ca²⁺ but is downstream of Src kinase. NR8383 macrophages were pretreated with (A) calcium chelator BAPTA-AM (10 μM), adenylyl cyclase inhibitor SQ22536 (10 μM), both BAPTA-AM and SQ22536 (10 μM each), or (B) Src kinase inhibitor PP2 and negative control PP3 for 30 min before the addition of 30/1 IgG-opsonized SRBCs. Cells were lysed and assayed for GTP-bound Rap1 as described in Materials and Methods. Total levels of Rap1 in cell lysates are shown (bottom blot). One blot representative of a total of four is shown; densitometric analysis was performed for each of the four Western blots as described in Materials and Methods.