Figure 2.

Determination of the CIP4 Binding Domain of COP1.

(A) Diagrams of wild-type and COP1 deletion constructs used for the assay. All of the deletion proteins were fused with a Flag tag and an HMK recognition site sequence (Yamamoto et al., 1998). The three protein–protein interaction domains and domain deletions are marked.

(B) The binding capacities of three versions of GST–CIP4 fusion proteins, and GST itself with full-length ³²P-labeled COP1.

(C) The binding capacities of the ³²P-labeled COP1 deletion proteins toward the long version of GST–CIP4 in the in vitro binding assay. The input COP1 probes are shown at left and the bound COP1 proteins are shown at right. Calculated molecular masses of the COP1 deletion proteins are shown at right in kilodaltons, and the deletion proteins are indicated by asterisks.

(D) The radioactivity of each band in (C) was quantified, and the ratios of the bound fractions relative to the total input were calculated.