Time course of TPO-induced MAPK phosphorylation. Bone marrow cells were prepared and stimulated as described in the legend to Fig 4. Cell lysates were subjected to Western blot analysis and probed with anti–double-phosphorylated ERK antibody (upper panels) and then stripped and reprobed with anti-ERK2 antibody (lower panels). The cultures contained (A) 14 ng/mL TPO alone or (B) TPO plus 10% (vol:vol) fetal calf serum or TPO plus 100 ng/mL SDF-1. The TPO-inducible bands found just above ERK1 were sometimes seen. They were not cross-reactive with anti-ERK1 antibody as the blot was stripped and reprobed. The origin of these bands is still unclear.