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. 2008 Jun;20(6):1437–1455. doi: 10.1105/tpc.108.058891

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Copyright © 2008, American Society of Plant Biologists

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Figure 4. — Evidence for a CUL4-DDB1-DCAF1 Complex in Arabidopsis.

(A) and (B) Association of DCAF1 with Flag-DDB1 in vivo. Total seedling protein extracts prepared from wild-type and 35S:Flag-DDB1 (Flag-DDB1) transgenic Arabidopsis were incubated with anti-Flag antibody-conjugated agarose (A) or with anti-DCAF1 antibody and Protein A sepharose (B). The immunoprecipitates (IP) and the total extracts (total) were subjected to immunoblot analysis with antibodies against Flag (for Flag-DDB1) and DCAF1.

(C) Association of DCAF1 with RUB-modified Flag-CUL4 in vivo. Total seedling protein extracts prepared from wild-type and 35S:Flag-CUL4 (Falg-CUL4) transgenic Arabidopsis plants were incubated with anti-DCAF1 antibody and Protein A sepharose. The immunoprecipitates (IP) and the total extracts (total) were subjected to immunoblot analysis with antibodies against Flag (for Flag-tagged CUL4 and CUL4-RUB) and DCAF1.

(D) Association of DCAF1-Flag with both DDB1 and CUL4 in vivo. Total seedling protein extracts prepared from wild-type and 35S:DCAF1-Flag (DCAF1-Flag) transgenic Arabidopsis were incubated with anti-Flag antibody-conjugated agarose. The immunoprecipitates (IP) and the total extracts (total) were subjected to immunoblot analysis with antibodies against Flag (for DCAF1-Flag), DDB1, and CUL4.

(E) DCAF1 does not associate with COP1 in vivo. Total seedling protein extracts prepared from wild-type Arabidopsis were incubated with anti-DCAF1 antibody and Protein A sepharose. Incubation with Protein A sepharose alone was used as mock. The immunoprecipitates (IP) and the total extracts (total) were subjected to immunoblot analysis with antibodies against DCAF1, DDB1, and COP1.

(F) DCAF1 does not associate with Flag-COP10 in vivo. Total seedling protein extracts prepared from 35S:Flag-COP10 (Flag-COP10) transgenic Arabidopsis were incubated with anti-DCAF1 antibody and Protein A sepharose. Incubation with Protein A sepharose alone was used as a mock. The immunoprecipitates (IP) and the total extract (total) were subjected to immunoblot analysis with antibodies against DCAF1, DDB1, and Flag (for Flag-COP10).

(G) Flag-COP10 does not associate with DCAF1 in vivo. Total seedling protein extracts prepared from wild-type and 35S:Flag-COP10 (Flag-COP10) transgenic Arabidopsis were incubated with anti-Flag antibody-conjugated agarose. The immunoprecipitates (IP) and total extracts (total) were subjected to immunoblot analysis with antibodies against DCAF1, DDB1, COP1, DET1, and Flag (for Flag-COP10). The asterisks mark the cross-reacting bands (to the left).