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. 2021 Apr 24;296:100707. doi: 10.1016/j.jbc.2021.100707

Figure 5.

Figure 5

CtIP weakened the binding of Drosha with DGCR8 and RNA substrates.A, coimmunoprecipitation of Drosha and DGCR8 was performed in wildtype HCT116 cells and CtIP-KO cells using anti-DGCR8 antibody. B, coimmunoprecipitation between GFP-Drosha and DGCR8 was performed in wildtype HCT116 cells and CtIP-KO cells expressing GFP-Drosha or empty vector using anti-GFP antibody. C, 293T cells cotransfected with Drosha-VN and DGCR8-VC, along with Flag-CtIP or control vector, fluorescent images were taken at 24 h after transfection. The scale bars represent 100 μm. The chart showed the cell fluorescence quantified by ImageJ. Data represent the means ± SD. The p value is indicated as ∗p < 0.05. D, RNA-ChIP analysis of association between Drosha and pri-miR302b was performed in wildtype HCT116 cells and CtIP-KO cells using anti-Drosha antibody and pri-miR302b primer sets. The ChIP value in wildtype cells was set as 1 for normalization. Data represent the means ± SD of three independent experiments. The p value is indicated as ∗∗p < 0.01. E, in vitro pri-miRNA processing assay was performed by incubating pri-miR302b substrate with immunoprecipitated Flag–Drosha complex in the presence or absence of recombinant GST-CtIP protein. ChIP, chromatin immunoprecipitation; CtIP, C-terminal-binding protein–interacting protein; DGCR8, DiGeorge syndrome critical region gene 8; HCT116, human colon cancer cell line; pri-miRNA, miRNA primary transcripts; VC, Venus C-terminal fragment; VN, Venus N-terminal fragment.