Kinetics of ERK2 activation in eosinophils stimulated with eotaxin.
Eosinophils were incubated with medium or stimulated with eotaxin (10−8 mol/L) for 0.5, 1, 2.5, 5, 15, or 30 minutes. Cytosolic extracts were immunoprecipitated with the anti-ERK2 antibody. (A) Tyrosine phosphorylation of ERK2 was detected by Western blotting (WB) with the anti-phosphotyrosine (α-PY) antibody. (B) Concomitant bandshifting was detected by reprobing the membrane with the anti-ERK2 antibody. This also verified that the tyrosine-phosphorylated band was ERK2 (n = 3). (C) ERK2 immunoprecipitates were assayed for kinase activity using MBP as the substrate. The kinase activity following eotaxin stimulation paralleled the tyrosine phosphorylation profile of ERK2. The numbers on the left side of the panels indicate the position of the molecular weight markers.
