Figure 4.

GAS6-AS1 interacted with transcription factor E2F1

(A) RNA expression was measured by qRT-PCR after nuclear and cytosolic separation. U6 small nuclear RNA (U6) was used as a nucleus marker, and glyceraldehyde-phosphate dehydrogenase (GAPDH) was used as a cytosol marker. (B) Transcription factors that are predicted to interact with GAS6-AS1 by LncMAP. (C) RIP experiments were performed in A549 cells with E2F1 antibody, and IgG was used as an internal reference. (D) Schematic diagram of RNA pull-down assay. (E) Lysates from A549 cells were used for RNA pull-down with biotinylated GAS6-AS1 transcript or antisense transcript. Western blotting demonstrated the specific association of GAS6-AS1 with E2F1. (F and G) qRT-PCR (F) and western blotting (G) were performed to detect the mRNA and protein expression of E2F1 after overexpression of GAS6-AS1. (H and I) The predicted interaction profile (H) and binding region (I) of GAS6-AS1 with E2F1 are shown. (J) The secondary structure of GAS6-AS1 in AnnoLnc is presented. (K) A series of GAS6-AS1 deletion mapping fragments were constructed, and RNA pull-down assays were performed. (L) Western blotting analysis was performed with the proteins retrieved from pull-down assays, and input was used as a positive control.