Stat activation in tumors and cell lines. (A-B) Immunoprecipitation of Stat1 and Stat3. Cell lines were either untreated (–), or stimulated with IFN-α (α), or stimulated with IFN-γ (γ) for 1 hour. Thereafter the cells were lysed and the Stat proteins immunoprecipitated. The membranes were first blotted with an antibody directed against phosphotyrosine (PTYR) and then destripped and reprobed with antisera directed against Stat1 (A) or Stat3 (B). (C) Gel shift analysis for Stat1 and Stat3 in the cell lines treated as described above for panels A and B. The specificity of the Stat1-containing complexes was proven by using Abelson-transformed B-cell lines derived from Stat1-deficient fetal livers (not shown). (D) Five tumors derived from Ab-MuLV–transformed Jak1(–/–) cells (first 5 lanes) and 4 tumors from Ab-MuLV–transformed Jak1(+/–) cells (last 4 lanes) were analyzed by gel shift for Stat1 and Stat3 activation. (E) Gel shift analysis for Stat1 and Stat3 activation in cell lines and the corresponding primary tumors. Samples 1 and 4 are deficient for Jak1; samples 2 and 3 are derived from Jak1(+/–) fetal livers. (F) Stat5 and Stat6 activation in Jak1(+/–) and Jak1(–/–) cell lines. Gel shift analysis and supershift analysis with antibodies directed against Stat5 (5) and Stat6 (6) shows activation of Stat5 but not Stat6. An unrelated antibody (C) was used as additional control. Slash indicates that no antibody was added.