FIGURE 1.

Association of the CCR4-NOT polypeptides in a stable complex. (A) Detection of NOT1 with a monoclonal antibody. Indicated amounts of an extract from 0- to 2-h-old Drosophila embryos were separated on a 6% SDS–polyacrylamide gel, blotted to a PVDF membrane, and probed with the monoclonal antibody CISS 2G5. Sizes of marker proteins are indicated. (B) Western blot analysis of NOT1 immunoprecipitation from embryo extract. The monoclonal NOT1 antibody CISS 2G5 and a monoclonal rat antibody against human hnRNP K were used for immunoprecipitation (lanes labeled N1 and c, respectively). Equal amounts of input and flow-through were loaded. Bound proteins were eluted with the peptide used for immunization. A polyclonal antibody (CR#1) directed against the same peptide was used for detection of NOT1. Polyclonal antibodies for detection of the other subunits are indicated on the left. Note that the CCR4 antibody recognizes a doublet in embryo extracts. Only the lower band (arrowhead) is depleted by NOT1 immunoprecipitation and enriched in the peptide eluate. The identity of the upper band is unknown.