Orthovanadate did not activate ERK-1 activity, and ERK activity is required for proliferation.

(A) HCD57 cells were deprived of EPO and cultured 24 hours in 0 (lanes i and ii), 10 (lanes iii and iv) or 70 (lanes v and vi) μmol/L orthovanadate. The cells were either stimulated with 10 units EPO per milliliter (lanes ii, iv, and vi) or mock treated for 5 minutes (lanes i, iii, and v). ERK-1 was immunoprecipitated from the cell extracts; activity was determined by the phosphorylation of myelin basic protein in an in vitro kinase assay. (B) HCD57 cells were cultured in the presence or absence of EPO and in the presence or absence of 50 μmol/L PD98059, MEK inhibitor to block ERK activity. Cells were counted at 24, 48, 72, and 96 hours. Viability was determined using trypan blue exclusion. Error bars indicate the standard deviation of 4 determinations.