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. 2021 Apr 15;131(8):e146431. doi: 10.1172/JCI146431

Figure 4. ELNAT1 directly interacts with hnRNPA1.

Figure 4

(A) RNA pull-down assay of ELNAT1 in UM-UC-3 cells. (B) MS analysis of the proteins from the RNA pull-down assay. (C and D) RNA pull-down with nuclear extract (C) and Western blotting with purified recombinant hnRNPA1 (D) were performed to evaluate the interaction between ELNAT1 and hnRNPA1. (E) Immunofluorescence was performed to assess the colocalization of ELNAT1 and hnRNPA1 in UM-UC-3 and T24 cells. Scale bars: 5 μm. (F) RIP assay using anti-hnRNPA1 to assess the enrichment of ELNAT1 by hnRNPA1. IgG was used as a negative control; U1 was used as a nonspecific control. A 2-tailed Student’s t test was performed to determine statistical significance. (G and H) RNA pull-down assays using serial deletions of ELNAT1 were performed to evaluate the regions required for the binding of ELNAT1 and hnRNPA1. (I) Prediction for the stem-loop structures of hnRNPA1-binding sites in ELNAT1. (J) RIP assay after deletion of 610–680 nt of ELNAT1 in UM-UC-3 cells. A 2-tailed Student’s t test was used to assess statistical significance. Error bars show the SD of 3 independent experiments. **P < 0.01.