JNK and p38 activities are high in HCD57 cells in the presence of EPO and decrease on EPO withdrawal.

HCD57 cells were deprived EPO for up to 96 hours and samples were taken at 24-hour intervals. (A) Rabbit-anti-JNK-1 immunoprecipitates were subjected to an in vitro kinase assay using γ-³²P-ATP and GST-Jun fusion protein as a substrate and transferred to nitrocellulose. Following exposure to show the phosphorylated GST-Jun (upper arrow), the proteins were immunoblotted with goat anti-JNK-1 to visualize JNK proteins (lower arrow). (B) Whole cell lysates were immunoblotted with an anti-phospho-JNK antibody (upper panel). The blot was then stripped and reprobed with anti-JNK1 to ensure equal loading of proteins (arrow, lower panel). (C) Whole cell lysates were immunoblotted with an anti-phospho-p38 antibody (upper panel). The blot was then stripped and reprobed with anti-phospho-ERK antibody (middle panel) to detect phosphorylated ERK (arrow, middle panel) and p38 (lower panel) to ensure equal loading of proteins (arrow, lower panel). (D) HCD57(K) cells were deprived of EPO for the times indicated. Whole cell lysates were immunoblotted with anti-phospho-JNK antibody (top panel). Arrows indicated phosphorylated JNK-1 and -2 proteins. The blot was then stripped and reprobed with anti-phospho-p38 antibody to detect phosphorylated p38 (arrow, middle panel) and anti-JNK1 to ensure equal loading of proteins (arrow, lower panel).