Role of the negative feedback regulator USP18 for IFNAR dimerization and STAT recruitment. (a and b) USP18 constitutively binds to IFNAR2: (a) HeLa cells transfected with HaloTag-tagBFP-IFNAR2 (cyan channel) and EGFP-USP18 (green channel) after incubation of ^AT655IFNα2 (red channel). Cell boundaries are indicated by dotted lines. The squares indicate the analyzed sectional distribution of fluorescence intensity shown in b. Bars, 10 µm. (b) Contrast of USP18 before and after incubation of ^AT655IFNα2 compared with HaloTag-tagBFP-IFNAR2 (representative of three cells analyzed). (c and d) Ternary complex formation and USP18 binding to IFNAR2 are noncompetitive: U5A cells transfected with HaloTag-IFNAR1, SNAP-IFNAR2, and EGFP-USP18 (green channel) before and after addition of IFNα2 (red channel; representative of three cells analyzed). Cell boundaries are indicated by dotted lines. The rectangles indicate the analyzed sectional distribution of fluorescence intensity shown in d. Bars, 10 µm. (d) Intensity pattern in the area indicated in c. (e and f) The stability of constitutive STAT2 binding to IFNAR2 is affected by USP18: (e) STAT2-tagRFP (yellow channel) bound to micropatterned HaloTag-IFNAR2 in cells coexpressing USP18 was bleached with a 405-nm laser in the indicated area (dotted circles), and recovery was monitored. Bars, 5 µm. (f) Comparison of the fluorescence recovery of STAT2 bound to IFNAR2 in the absence and in the presence of ectopic USP18 (representative of 10 cells analyzed in the absence of USP18 and five cells analyzed in the presence of USP18). rel., relative.