Inhibition of Bcr-abl activity in CML cells stabilizes and up-regulates IFNAR1. (A) Cell surface levels of IFNAR1 in KT1 cells treated as indicated were analyzed by flow cytometry using anti-IFNAR1 AA3 antibody or isotype control antibody. (B) Total levels of IFNAR1 and β-actin in KT1 or HeLa cells treated with indicated doses of IM for 8 hours were analyzed by immunoblotting. Short exposure for IFNAR1 blot in HeLa cells also is provided. (C-D) KT1 or KU812 CML cells were incubated with IM (0.5μM) for 8 hours and harvested. Phosphorylation and total levels of IFNAR1 were analyzed by IFNAR1 immunoprecipitation (using anti-IFNAR1 EA12 antibody) followed by immunoblotting using anti–phospho-S535 antibody or anti-IFNAR1 GB8 antibody (as indicated). (E) Primary CML cells from a patient sample (623) were incubated with IM (0.5μM) for 8 hours and harvested. Phosphorylation and total levels of IFNAR1 were analyzed by IFNAR1 immunoprecipitation (using anti-IFNAR1 EA12 antibody) followed by immunoblotting using anti–phospho-S535 antibody or anti-IFNAR1 GB8 antibody (as indicated). Supernatants of the immunoprecipitation reactions were immunoblotted for β-actin to determine loading. (F) KT1 cells pretreated with IM (0.5μM) or vehicle for 8 hours were subjected to treatment with CHX (10μg/mL) for the indicated time points. Endogenous IFNAR1 was immunoprecipitated and levels of IFNAR1 were determined by immunoblotting. Amount of lysates was normalized to achieve comparable levels of IFNAR1 at time point 0. Levels of β-actin in reaction supernatants also were analyzed.