FIGURE 5.

Calcium is required for AA release but not ERK and p38 activation in response to serum and PMA. A, IMLF^+/+ were preincubated with 10 mm EGTA for 15 min and left unstimulated (US) or treated with serum for 10 min or PMA for 45 min. [³H]AA released into the medium was measured and presented as a percentage of total cellular radioactivity. B, IMLF^+/+ were preincubated with or without EGTA and stimulated with serum or PMA for the indicated times. Immunoblots were conducted using equal protein per lane and probed with phosphospecific antibodies against phospho-ERK and phospho-p38 or with antibody to total ERKs to determine sample loading. C, [³H]AA-labeled IMLF^-/- expressing either wild type ECFP-cPLA₂α, EYFP-cPLA₂αD43N or neither (uninfected) were stimulated with 10% serum (10 min) or 100 nm PMA (45 min), and [³H]AA release was compared with unstimulated cells. Wells with matching expression levels of cPLA₂α wild type and mutant (inset) determined by Western blot analysis were used for [³H]AA release determination. Results are representative of a minimum of three independent experiments conducted in duplicate. The release of [³H]AA by the mutant was significantly less (p < 0.05) than by wild type cPLA₂α (WT), as indicated (^*).