Fig. 2.

PKC participation in sPLA₂-IIA–induced activation of ERK, cPLA₂, and Ras. (A and C) 1321N1 cells were incubated with the indicated doses of GF109203X 30 minutes prior to stimulation with sPLA₂-IIA. Western blots of total lysates were incubated with either phospho-ERK (upper A) or actin (lower A) antibodies or cPLA₂ antibody (C). (B) 1321N1 cells were incubated with 1-μM PMA overnight or with 1-μM GF109203X for 30 minutes, prior to the addition of sPLA₂-IIA for 5 minutes. Western blots of the lysates were incubated either with phospho-ERK (upper) or with actin antibody (lower). Samples shown belong to the same experiment and were run simultaneously but not consecutively; therefore, a lane represents the gap. (D) 1321N1 cells were incubated with 1-μM PMA overnight and then with sPLA₂-IIA for the indicated times, and RasGTP was determined using the RBD pull-down assay. The first lane shows a sample of total lysate; the other lanes are Ras bound to the RBD, which represents RasGTP. In all cases, cells were serum starved overnight and the experiments shown are representative of the results obtained in at least 3 trials for each condition.