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. 2016 Aug 16;28(9):2043–2059. doi: 10.1105/tpc.16.00474

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© 2016 American Society of Plant Biologists. All rights reserved.

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Figure 6. — AGO4 Protein Contents.

(A) Immunodetection of AGO4 in cellular extracts of wild-type (No and Col), ddb2-2, and ddb2-3 mutant plants (*, cross-reacting signal). Coomassie blue staining of the blot is shown.

(B) Immunodetection of AGO4 and DDB2 in wild-type Col, dcl3, and ago4 mutant plants. Coomassie blue staining was used as loading control (*, cross-reacting signal).

(C) Immunoblot analysis of AGO4 content in chromatin extracts from wild-type (Col) and ddb2-3 mutant plants. Anti-histone H3 and anti-UGPase antibodies were used as controls for insoluble (chromatin) and soluble fractions, respectively. P, pellet (insoluble fraction); S, supernatant (soluble fraction). Signal intensity relative to H3 is indicated below each lane (*, cross-reacting signal).

(D) Immunoblot analysis of DDB2 protein content in chromatin extracts from wild-type (Col) and ago4 mutant plants. Anti-histone H3 antibody was used as control for chromatin (insoluble fraction) and anti-UGPase antibody was used as control for soluble fraction. P, pellet (insoluble fraction); S, supernatant (soluble fraction). Signal intensity relative to H3 is mentioned below each lane.

(E) ChIP of AGO4 at three hyper-DMRs (TE) and two hypo-DMRs (TE) in wild-type (Col) and ddb2-3 plants using anti-AGO4 antibody. As negative controls, ago4 plants were used as well as Actin2. Data are presented as enrichment of AGO4 signal (±sd) and are representative of three biological replicates.

(F) In vivo pull-down of AGO4 with DDB2 protein. No, ddb2-2 DDB2-FLAG, and ddb2-2 DDB2^K314E-FLAG-expressing plants were used for immunoprecipitation assays using anti-FLAG. Signal intensity relative to control is mentioned below each lane.

(G) Amount of DDB2 and AGO4 in chromatin, as analyzed by immunodetection in ddb2-2 DDB2-FLAG, ddb2-2 DDB2^K314E-FLAG, and ddb2-2 DDB2^W261F-FLAG-expressing plants. Anti-histone H3 antibody was used as control for chromatin (insoluble fraction) and anti-UGPase antibody was used as control for soluble fraction. P, pellet (insoluble fraction); S, supernatant (soluble fraction). Signal intensity relative to H3 is mentioned below each lane (*, cross-reacting signal).