Figure 4.

Fendrr binds to STAT2.A, silver staining of proteins bound to Fendrr. The RNA pull-down was performed with AML12 cell lysates. A specific band was identified as STAT2 by mass spectrometry. B, the interaction between Fendrr and STAT2 was confirmed by RNA pull-down and Western blot. The biotin-labeled Fendrr and antisense control were mixed with the extracts from AML12 cells, and then incubated with streptavidin agarose beads. The retrieved proteins were assayed by Western blot. C, RNA immunoprecipitation (RIP) assay revealed that Fendrr interacts with STAT2. Whole-cell lysates of AML12 cells were immunoprecipitated with STAT2 antibody or IgG. Western blot analysis confirmed IP of endogenous STAT2 from AML12 cells (left). qPCR analysis for Fendrr and albumin mRNA in the anti-STAT2 or IgG immunoprecipitates revealed the binding of Fendrr to STAT2 (right). The results were shown as the copy number per μg RNA. D, subcellular fractionation and quantitative assay of Fendrr copy numbers in the cytoplasm and nucleus of AML12-Fendrr cells. About 1 × 10⁶ AML12-Fendrr cells were collected for subcellular fractionation and RNA purification. The results were shown as the copy number of Fendrr per 10⁶ cells. E, Fendrr promotes the enrichment of STAT2 in the nuclei of hepatocytes. AML12-Fendrr cells were stained for STAT2. Nuclei were visualized using DAPI staining (blue). The scale bar represents 10 μm. F, nuclear fractionation and Western blot assay of STAT2 in the nucleus (left). Relative protein level was calculated by band intensity against histone 3 (right). Results from at least three independent experiments are shown as mean ± SD. ∗∗∗∗p < 0.0001, and ns, no significant change. DAPI, 4,6-diamidino-2-phenylindole; IgG, immunoglobulin G; IP, immunoprecipitation; qPCR, quantitative PCR.