Fig. 3

LINC00623 binds to NAT10. a GO-CC (cellular component) analysis predicting the potential proteins binding to LINC00623. b Western blotting was performed to evaluate the specific association of NAT10 with biotinylated LINC00623. Lysates of BxPC-3 and PANC-1 cells were harvested for RNA pulldown assays. LINC00623 antisense RNA was used as the negative control. c A RNA-binding protein immunoprecipitation (RIP) assay was performed using an antibody against NAT10. RT-PCR was used to detect LINC00623 enrichment. IgG was used as the isotype control. d Immunofluorescence (IF) staining showed that LINC00623 (red) was colocalized with NAT10 (green) in the nucleus. Scale bar = 10 μm. e Western blotting of NAT10 in samples precipitated by biotinylated full-length LINC00623 (FL) or LINC00623 truncations (Δ1: 1–699 bp; Δ2: 700–1200 bp; Δ3: 1201–1800 bp; Δ4: 1801–2500 bp; Δ5: 2501–3000 bp; Δ6: 3001–3607 bp). FL LINC00623 was used as the positive control. f Flag-RIP assay for LINC00623 showing its fold enrichment in cells transiently transfected with plasmids containing FLAG-tagged full-length and truncated NAT10 constructs (Del1: 1–280 aa; Del2: 281–488 aa; Del3: 558–753 aa; Del4: 753–1052 aa). IgG-RIP was used as the internal control. The values are expressed as the mean ± SD of three independent experiments