JAK/STAT activation in response to EPO.

DP16.1/p53ts cells were cultured at 37°C or at 32°C for 12 hours, then treated with EPO for 15 minutes and lysed immediately for immunoprecipitation and Western blotting. (A) JAK1, JAK2, and STAT3 proteins were immunoprecipitated with specific antibodies and analyzed by Western blotting with antiphosphotyrosine antibodies. The same blots were stripped and reprobed with JAK1, JAK2, or STAT3 antibodies. (B) DP16.1/p53ts cells cultured as above were lysed and subjected to SDS-PAGE and Western blotting with phospho-specific antibodies against STAT1 or STAT5 (Tyr694). An extract prepared from BAF3 cells expressing a Tel-Jak2 fusion protein in which STAT1 is phosphorylated served as a positive control for the phospho-STAT1 antibody. (C) EMSA for STAT5 DNA-binding activity. DP16.1/p53ts cells were cultured at 37°C (lanes 1, 4, 7, and 10) or 32°C for 12 hours (lanes 2, 3, 5, 6, 8, 9, 11, and 12). Nuclear extracts were prepared and incubated with a radiolabeled oligonucleotide probe derived from the β-caseinpromoter. An antibody against STAT5 was included in the binding reaction in lanes 4 to 6. For competition analysis, a 50-fold molar excess of unlabeled β-casein oligonucleotide (lanes 7-9) or an unrelated DUB-1 promoter oligonucleotide (lanes 10-12) was added to the binding reaction. The DNA-binding complexes are indicated by the bottom arrow and the supershifted complexes are indicated by the top arrow.