Figure 6.
A high-molecular weight complex containing DET1 coelutes transiently with DDB2 following UV-C exposure. (A, B) DET1 and DDB2 fractionate in different protein complexes under normal light conditions. (A) Size-exclusion chromatography analysis of DET1 complex in protein extracts from wild-type (Nossen) and ddb2-2 mutant seedlings. Fractions were analysed by immunoblot using DET1 and DDB2 antibodies. Arrows indicate elution of molecular-weight standards in the same conditions. (B) Size-exclusion chromatography analysis of DDB2 complex in protein extracts from wild-type (Col-0) and det1-1 mutant. (C) A DET1 high-molecular weight complex is transiently detected upon UV-C irradiation. Experiment was performed as in (A) except that seedlings were exposed to UV-C (900 J/m2) and harvested after 30 or 60 min for protein extraction and size-exclusion chromatography. The inset shows DET1 protein content in input samples before chromatography separation. (D) Size-exclusion chromatography of protein extracts from wild-type Nossen and ddb2-2 mutant plants irradiated at 900 J/m2 UV-C and harvested 30 min after UV exposure. The inset shows DET1 input content in wild-type, det1-1 and ddb2-2 seedlings harvested before (−UV) or 30 min after UV-C exposure (+UV). Ponceau staining is used as control for equal fractionation of the two samples. Arrows indicate elution of molecular-weight standards in the same conditions. (E) Side-by-side analysis of fractions 5 and 6 containing DET1 UV-induced HMW complex. Fractions 5 and 6 (50 μl) from size-exclusion separation of wild-type Nossen, ddb2-2 and det1-1 extracts were analysed by immunoblot using a DET1 antibody. The same blot was probed with anti-RbCL antibody as a loading control. (F) Protein content of DET1, DDB2 and CUL4 in det1-1 mutant and wild type before and 30 min after UV-C exposure. Whole cell protein extracts (40 μg) were separated on 10% SDS–PAGE and analysed with the indicated antibodies.