Fig. 7.
Fig. 7. EPO-dependent STAT5 DNA binding is activated in several EPO-R tyrosine mutants. / Nuclear extracts were prepared from Ba/F3–EPO-R (lanes 1-6), Ba/F3 EPO-R Y1Y2Y4Y8 (lanes 7-10), Ba/F3 Y1Y2Y4 (lanes 11-14), Ba/F3 Y1Y2 (lanes 15-18), and Ba/F3 EPO-R F8 (lanes 19-21) cells stimulated in the presence or absence of IL-3 (I) or EPO (E). Electrophoretic mobility shift assays were performed as described in “Materials and methods.” Complexes were resolved on a 5% native polyacrylamide gel. The specificity of DNA binding was determined by the addition of unlabeled β-casein oligonucleotide (S), a nonspecific oligonucleotide from the DUB-1 promoter (N), and by incubation with a peptide-specific STAT5 antibody. Complexes were analyzed via PhosphorImager detection.

EPO-dependent STAT5 DNA binding is activated in several EPO-R tyrosine mutants.

Nuclear extracts were prepared from Ba/F3–EPO-R (lanes 1-6), Ba/F3 EPO-R Y1Y2Y4Y8 (lanes 7-10), Ba/F3 Y1Y2Y4 (lanes 11-14), Ba/F3 Y1Y2 (lanes 15-18), and Ba/F3 EPO-R F8 (lanes 19-21) cells stimulated in the presence or absence of IL-3 (I) or EPO (E). Electrophoretic mobility shift assays were performed as described in “Materials and methods.” Complexes were resolved on a 5% native polyacrylamide gel. The specificity of DNA binding was determined by the addition of unlabeled β-casein oligonucleotide (S), a nonspecific oligonucleotide from the DUB-1 promoter (N), and by incubation with a peptide-specific STAT5 antibody. Complexes were analyzed via PhosphorImager detection.

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