ERK1 and ERK2 are constitutively phosphorylated in HS2 cells.

(A) 663 HS1 cells were either grown in the presence of 2 U/mL Epo and 10% serum or starved in Epo and serum for 4 hours. 606 HS2 cells were either grown in the presence of 10% serum or starved in serum for 4 hours or starved and then stimulated with 10 U/mL Epo and serum for 30 minutes. ERK1 was immunoprecipitated from cell extracts and an in vitro kinase assay was performed using the MBP peptide as substrate. The means and standard deviations were determined from 3 experiments. (B) 663 HS1 cells were either grown in the presence of 2 U/mL Epo and 10% serum or starved in Epo and serum for 4 hours before being stimulated with 10 U/mL Epo and/or 10% serum. A quantity of μM of LY294002 or 20 μM of U0126 was added for the last hour of starvation. (C) 606 HS2 cells were either grown in the presence or absence of 2 U/mL Epo and 10% serum or starved in Epo and serum for 4 hours before being stimulated with 10 U/mL Epo and/or 10% serum. They were treated with 10 μM LY294002 or 20 μM U0126 for the last hour of starvation. Cell lysates were immunoblotted with anti–phospho-ERK1/2 (B, C upper panels) and anti-ERK1/2 (B, C lower panels) antibodies. (D) Protein extracts of 622, 921, and 931 unstimulated HS2 cell lines were immunoblotted with anti–phospho-ERK1/2 (upper panel) and anti-ERK1/2 (lower panel) antibodies. The positions of phosphorylated ERK1 and ERK2 (P-ERK1, P-ERK2) and ERK1 and ERK2 were indicated.