Fig. 5

circEHD2 direct interacts with YWHAH. A, RNA pull-down assay was applied in RCC cells to identify the proteins that interacted with circEHD2. B, The silver staining image of RNA pull-down with circEHD2 probe in OSRC-2 cells. C, Mass spectrometry (MS) analysis of the proteins interacted with circEHD2 via RNA pull-down assay. D, qRT–PCR analysis confirmed that the circEHD2 probe could specifically enrich circEHD2 in OSRC-2 cells by RNA pull-down assay. E, Western blot assay in circEHD2 pull-down proteins confirmed the interaction between circEHD2 and YWHAN. F, The subcellular co-localization of circEHD2 and YWHAH in RCC cells was measured by fluorescence staining assay. Scale bars: 10 μm. G, RIP assay in OSRC-2 cells confirmed that circEHD2 could be enriched by YWHAH. IgG was used as a negative control. H, The three-dimensional structure showed circEHD2 could be bound to YWHAH. I, Serial deletions of circEHD2 were used in RNA pull-down assays to confirm the regions of circEHD2 that were required for YWHAH. J, qRT–PCR analysis of the level of circEHD2 when mutated the binding sites of circEHD2. K, RIP assay was performed in OSRC-2 cells after mutating the 201-252-nt region of circEHD2. L, The knockdown efficiency of YWHAH in RCC cells measured by qRT-PCR. M, qRT–PCR analysis of the level of SOX9 when knockdown of YWHAH in RCC cells. N, Western blot analysis of the expression of SOX9 when downregulation of YWHAH in OSRC-2 cells. O, qRT–PCR analysis of the level of SOX9 in RCC cells after mutating the 201-252-nt region of circEHD2. P, Western blot assay revealed the level of SOX9 in OSRC-2 cells after mutating the 201-252-nt region of circEHD2. Error bars, standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001