Sustained activation of STAT5 is necessary for the induction of β-casein transcription. (A and B) Blocking sustained STAT5 activation with AG490 inhibited β-casein transcription. After EpH4 cells on polyHEMA were incubated with Prl and 2% lrECM for 8 h, AG490 or DMSO vehicle alone were added to the media at different concentrations. (A) Western blot analysis of STAT5 reactivation in control and AG490-treated cells. (B) Quantification of β-casein mRNA levels in control and AG490-treated cells by real-time RT-PCR. (C and D) Interrupting the sustained STAT5 reactivation with AG490 significantly inhibited β-casein transcription. EpH4 cells on polyHEMA were incubated with Prl and 2% lrECM for 24 h to induce the sustained STAT5 activation; the cells were then treated with 25 μM AG490 or DMSO vehicle alone for another 24 h. (C) Western blot analysis of the sustained STAT5 activation in control and AG490-treated cells. (D) Real-time RT-PCR measuring β-casein mRNA levels after the sustained STAT5 activation was interrupted. The bar graph represents the mean ± SEM.