Inhibition of Lyn activity increases Erk1/2 phosphorylation and activation in BaF3/Mpl cells. BaF3/Mpl cells were starved overnight, stimulated with 15 ng/mL TPO in the presence or absence of 2 μM PP2, and lysates prepared after the indicated times. (A) Samples normalized for protein concentration were separated by SDS-10% PAGE and analyzed by immunoblotting using an antibody specific for dually phosphorylated Erk1/2 (p42/p44). Blots were stripped and reprobed with Erk1/2-specific antibody to ensure equal loading. (B) Erk1/2 kinase activity was assayed by using Elk-1 as a substrate. Elk-1 phosphorylation at S383 was detected by Western blotting using a phospho-specific Elk-1 antibody. The increase in Elk-1 phosphorylation was quantitated by densitometry and expressed as fold change (in bar graph) and is representative of 2 separate experiments.