Withdrawal of EPO produces activation of JNKs and p38 MAP kinase preceding apoptosis of HCD-57 cells. HCD-57 cells (2 × 10⁷/mL) were incubated in EPO-free medium for the indicated periods of time and then either were labeled with fluorescein-dUTP for flow cytometry analyses (A) or were lysed in buffer A for JNK and p38 MAPK activation assays (B and C) as described in Materials and Methods. (A) Flow cytometry analyses of apoptotic HCD-57 cells. The left peak represents normal growing cells, whereas the right peak corresponds to apoptotic cells, and the percentage of cells that undergo apoptosis is indicated. (B and C; top 4 panels) Cell extracts (20 μg) were resolved on 10% SDS-PAGE, transferred to PVDF membranes, and probed with phospho-specific and regular antibodies against ERK1/2, JNK1/2, p38 MAPK, and MKK3/6 as indicated. (B and C; bottom panels) Cell extracts (containing 250 μg of total proteins) were incubated with an N-terminal c-Jun (1-89) fusion protein bound to glutathione Sepharose beads for JNK kinase activity assay or with anti-p38 MAP kinase antibody for p38 MAPK activity assays with ATF-2 as a substrate. Phosphorylation of c-Jun and ATF-2 was determined by Western blotting with the phospho-specific c-Jun antibody and ³²P autoradiography, respectively.