Induction of CIS inhibits STAT5 activity in Ba/F3 cells. (A) After BF-CIS-ER cells were cultured in the absence of growth factor for 0, 2, 4, and 6 hours in the presence or absence of 500 nmol/L Dex, cell extracts were prepared and CIS content was measured by immunoblotting. (B) After 6 hours starvation in the presence or absence of Dex, cells were stimulated with the indicated concentrations of EPO or IL-3 for 20 minutes. Expression of OSM, c-myc, and cdk4 was measured by Northern blotting. (C) After starvation for 6 hours, cells were stimulated with 10 U/mL EPO or 10 ng/mL IL-3, and STAT5 was immunoprecipitated, resolved by SDS-PAGE, and tyrosine-phosphorylation was detected by immunoblotting with anti-PY. The STAT5 content was checked by reprobing the membrane with anti-STAT5.