hCCR4 interacts with hCAF1 in vitro. Bacterially-expressed and purified GST-hCAF1 and GST (control) proteins were subjected to SDS/PAGE, transferred from the gel onto a polyvinylidene difluoride membrane, and probed with in vitro synthesized 35S-methionine-labeled hCCR4 (left) or luciferase (control, right) proteins (see Materials and Methods). Autoradiography of the blots demonstrates specific binding of hCCR4 to GST-hCAF1 (57 kDa, arrow), not observed with GST (27 kDa; position of the protein, as determined by a coomassie blue stained gel run in parallel, indicated by an arrowhead). Molecular size markers (in kilodaltons) are indicated. The negative imprints observed in the luciferase control are a common feature associated with reduction of background hybridization to the filter at locations of high protein concentrations.