Figure 4.

Down-regulation of IFNAR1 in cells that express Bcr-abl does not require CK1 or priming phosphorylation of IFNAR1. (A) HeLa cells transfected with Bcr-abl or treated with TG (1μM for 30 minutes) also were treated with CK1 inhibitor D4476 (400nM) and harvested. Phosphorylation and levels of endogenous IFNAR1 and expression of Bcr-abl was analyzed as described in Figure 3. (B) HeLa cells expressing FLAG-tagged IFNAR1 (wild type or IFNAR1^S532A or IFNAR1^S535A mutants) were cotransfected with either empty vector or vector expressing Bcr-abl. Total levels of FLAG-IFNAR1 and Bcr-abl were assessed by immunoblotting using the indicated antibodies. (C) Human fibrosarcoma 11.1-derivative cells expressing either wild-type TYK2 (WT) or kinase dead TYK2 (KR) were transfected with empty vector or vector expressing Bcr-abl. IFNAR1 was immunoprecipitated, and total IFNAR1 was assessed by immunoblotting. Supernatants of the immunoprecipitation reactions were immunoblotted for β-actin to determine loading. (D) HeLa cells transfected with Bcr-abl as indicated were treated with JAK inhibitor 1 (0.5μM for 24 hours). Levels of IFNAR1 after treatment was determined by immunoprecipitation and immunoblotting. Loading of the immunoprecipitation mixture was determined by immunoblotting the supernatants for β-actin. (E) KT1 cells were treated with IM (0.5μM) or N-acetyl cysteine (NAC; 1 or 3 μg/mL). Levels of IFNAR1 after treatment was determined by immunoprecipitation and immunoblotting. Loading of the immunoprecipitation mixture was determined by immunoblotting the supernatants for β-actin. (F) KT1 cells were treated with CID755673 (20μM) for the indicated time points. IFNAR1 was immunoprecipitated, and phosphorylated Ser535 (pS535) and total IFNAR1 was assessed by Western blotting. (G) KT1 cells were treated with CID755673 (20μM) for 6 hours and then treated with IFNα (250 IU/mL for 30 minutes). Levels of p-STAT1 and total STAT1 in whole cell lysates (WCL) were determined by immunoblotting using the indicated antibodies.