Fig. 2.

Time course of JNK activation and its relationship to depression of [Ca²⁺]_i by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-AM. A: effect of BAPTA-AM on [Ca²⁺]_i and JNK measured by an in vitro kinase assay. Cells were treated and analyzed as in Fig. 1A, except that 10 μM BAPTA-AM replaced A-23187. B: effect of BAPTA-AM on JNK activity in intact cells measured as amount of phosphorylated form of JNK as well as its substrates. Total cell lysates containing 15 μg protein were resolved by 10% SDS-PAGE, electroblotted onto polyvinylidene difluoride membranes, and immunostained with antibodies against the phosphorylated forms of the indicated proteins as described in MATERIALS AND METHODS. Equal protein loading was confirmed by staining the membrane with Ponceau Red (data not shown). Left lane: no stimulation. Right lane: stimulation for 30 min with 10 μM BAPTA-AM. *p46JNK, phosphorylated (Thr183/Tyr185) JNK, the p54JNK is phosphorylated identically as p46JNK (data not shown); *c-Jun, phosphorylated (Ser63) c-Jun; *ATF-2, phosphorylated (Thr71) ATF-2. The figure is representative of results from 3 independent experiments.