Two-hybrid assay for in vivo interaction of hCCR4 with hCAF1 and hCALIF/hPOP2. (A) schematic representation of the reporter and fusion protein expression vectors, and rationale of the two-hybrid assay. Coding sequences of hCCR4, hCCR4 deleted for the LRR (hCCR4(ΔLRR)) and m.nocturnin are inserted in-frame with the GAL4 binding domain. The hCAF1 and hPOP2 coding sequences (or no sequence in the pVP16-none vector) are inserted in-frame with the VP16 transactivation domain. The pG4-TK-Luc reporter plamid contains six GAL4 binding elements, upstream of the thymidine kinase minimal promoter (TK) region fused to the luciferase reporter gene (Luc). An interaction between the GAL4 and VP16 fusion proteins should result in an increase in luciferase expression. (B) hCCR4 interacts with hCAF1 and hPOP2, in a LRR-dependent manner. Hela cells were cotransfected with a combination of expression vectors, as indicated by crosses. Luciferase activities were measured two days post transfection (see Materials and Methods) and are expressed as the fold increase over the luciferase activity of the reporter vector alone. Bars indicate standard deviation of the mean for at least three independent transfections.