JNK and p38 are activated on EPO addition in HCD57 cells.
HCD57(R) cells were deprived of EPO for 18 hours and then incubated in EPO for the times indicated. (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. Arrow indicates phosphorylated GST-Jun. (B) Whole cell lysates were immunoblotted with an anti-phospho-JNK antibody (upper panel). Asterisk indicates phosphorylated ERK2 protein that cross-reacts with the phospho-JNK antibody. The blot was then stripped and reprobed with anti-phospho-ERK antibody (middle panel) to detect phosphorylated ERK-1 and -2 (arrows, middle panel) and anti-JNK1 (lower panel) 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-ERK-1 (lower panel) to ensure equal loading of proteins (arrow, lower panel). (D) ERK and JNK are not activated in a PI 3-kinase dependent manner in HCD57 cells. HCD57(R) cells were incubated for 24 hours in the presence of EPO alone (lane 1), EPO + DMSO (lane 3) or EPO + 50 μmol/L or 100 μmol/L LY294002 (lanes 2 and 4). Whole cell lysates were immunoblotted with an anti-phospho-JNK antibody (i). The blot was then stripped and reprobed with anti-phospho-ERK (ii, arrow), anti-phospho-AKT antibody to detect phosphorylated AKT (iii, arrow), and anti-JNK1 to ensure equal loading of proteins (iv, arrow).
