Maeda Mineko
Biological Science, Osaka University, Toyonaka, Japan.
Methods Mol Biol. 2006;346:469-78. doi: 10.1385/1-59745-144-4:469.
Intercellular signaling mediated by cAMP plays a pivotal role in coordinating cell movement into aggregates at the early stage of Dictyostelium development when the extracellular level of cAMP periodically changes at 6- to 7-min intervals. We have shown that MAP kinase ERK2 is activated via the cAMP receptor CAR1 in phase with this periodic change. This was revealed by assessing the level of ERK2 activation with immunoblots using two kinds of antibodies, commercially available anti-phospho-p44/p42 MAP-kinase antibody and anti-Dictyostelium ERK2 antibody. In this chapter, we describe the methods we have used to assess the level of activated ERK2 and partial involvement of G protein in cAMP-induced ERK2 activation.
在盘基网柄菌发育的早期阶段，当细胞外cAMP水平以6至7分钟的间隔周期性变化时，由cAMP介导的细胞间信号传导在协调细胞移动形成聚集体方面起着关键作用。我们已经表明，丝裂原活化蛋白激酶ERK2在此周期性变化的同一阶段通过cAMP受体CAR1被激活。这是通过使用两种抗体的免疫印迹法评估ERK2的激活水平而揭示的，这两种抗体分别是市售的抗磷酸化p44/p42丝裂原活化蛋白激酶抗体和抗盘基网柄菌ERK2抗体。在本章中，我们描述了我们用于评估活化ERK2水平以及G蛋白在cAMP诱导的ERK2激活中的部分作用的方法。
