Concentration-response of ERK2 and p38 activation in eotaxin-stimulated eosinophils.

Eosinophils were stimulated with various concentrations of eotaxin for 1 minute. For panels A, B, and C, cytosolic extracts were immunoprecipitated with the anti-ERK2 antibody. Tyrosine phosphorylation of ERK2 was detected by Western blotting with the anti-phosphotyrosine (anti-PY) antibody (A). Concomitant bandshifting was detected by reprobing the membrane with the anti-ERK2 antibody (B). This also verified that the tyrosine-phosphorylated band was ERK2 (n = 3). Further, the ERK2 immunoprecipitates were studied for kinase activity using MBP as the ERK2 substrate (C). For detection of p38 activation, cytosolic extracts underwent Western blotting with antibodies against dual threonine-tyrosine–phosphorylated p38 (α-PT/PY-p38) (D). The activation of both ERK2 and p38 by eotaxin was concentration-dependent.