Figure 5.
Chelation of cytosolic calcium with BAPTA blocks FCCP-stimulated phosphorylation and activation of ERK1 and ERK2. (A) Fura-2-loaded PC12 cells were monitored for basal [Ca2+]i. Cell medium was replaced at 5 min with the same medium containing 10 μM BAPTA-AM. At 15 min after the BAPTA addition, 30 μM FCCP in the presence of BAPTA was added. Data are representative of nine separate measurements. (B) PC12 cells were treated with either 10 μM BAPTA-AM, 0.5 mM EGTA, 10 mM caffeine alone for 15 min, or followed by 30 μM FCCP treatment for 1 hr. As a control, ionomycin (10 μM) was incubated with the cells for 15 min. The blot is representative of three separate experiments. (C) PC12 cells were pretreated with 10 μM BAPTA for 15 min followed by the addition of 30 μM FCCP for 1 hr. ERK1 and ERK2 kinase activity was assayed in phospho-ERK1+2-specific immunoprecipitates. Data are the means and SD of three separate experiments.