Role of PKD2 in Bcr-abl–induced IFNAR1 down-regulation and inhibition of IFNα signaling. (A) HeLa cells stably transduced with control shRNA or shPKD2 were transfected with either Bcr-abl or empty vector with or without cotransfection of shRNA-insensitive GST-PKD2 or GST-PKD2Y438F. Endogenous IFNAR1 was purified by immunoprecipitation and analyzed by immunoblotting using the indicated antibodies. Whole cell lysates (WCL) from these cells also were analyzed by immunoblotting for levels of PKD2, Bcr-abl, and β-actin. (B) HeLa cells stably transduced with control shRNA or shPKD2 and transfected with Bcr-abl/empty vector with or without shRNA-insensitive GST-PKD2 or GST-PKD2Y438F (as described in panel A) were treated with IFNα (250 IU/mL for 30 minutes). Activation and levels of STAT1 as well the levels of Bcr-abl, PKD2, and β-actin were analyzed by immunoblotting. (C) KT1 cells were transfected with control shRNA (shCON) or shRNA against PKD2 (shPKD2) and cotransfected with plasmid encoding hygromycin resistance. After 48 hours of selection in hygromycin, interferon signaling was investigated by immunoblotting the WCL for p-STAT1 and STAT1 after treatment with IFNα (250 IU/mL for 30 minutes).
