Figure 3.

AtCSN2 Is a Subunit of the COP9 Signalosome.

(A) Gel filtration analysis of AtCSN2 and comparison with the elution profiles of two other CSN subunits, AtCSN4 and AtCSN5. Total soluble protein extracts from 5-day-old light-grown Columbia seedlings were separated by gel filtration and subjected to immunoblot analysis with antibodies to AtCSN2 (top gel; the dot indicates a cross-reacting protein), AtCSN4 (middle gel), and AtCSN5 (bottom gel). Lane T indicates the total extract; lanes 1 to 25 indicate the gel filtration elution fractions. Numbers below the arrowheads indicate the positions of molecular mass markers (kD).

(B) AtCSN2 coimmunoprecipitates with other subunits of the COP9 signalosome. Total soluble protein extracts were mixed with AtCSN2 antibodies coupled to protein A beads (see Methods), and the immunoprecipitates (IP) were separated by SDS-PAGE. Antibodies used for immunoprecipitation are indicated at top, and antibodies used in the immunoblots are listed at right. PreI represents the preimmune serum from the same rabbit before immunization.

(C) AtCSN2 coimmunoprecipitates with subunits of SCF^TIR1. SCF complexes and their associated proteins were immunoprecipitated from total Arabidopsis protein extracts with AtCUL1 antibodies and subjected to immunoblot analysis with the antibodies listed at right. TBP antibodies were used to verify the specificity of the interactions.