Fig. 3

The metastasis-associated protein RAN was identified as the direct interacting protein of LINC00858 in GC. A Expression of LINC00858 in the cytoplasm and nucleus was detected via qPCR. B Diagram illustrating steps to identify interacting proteins. C Representative gels of immunoprecipitated proteins. D In vitro-synthesized LINC00858 was incubated with protein lysates from HGC27 and MKN74 cells. RAN protein was pulled down by biotin-labeled LINC00858 but not LINC00858 antisense RNA. E In vitro-synthesized LINC00858 was incubated with purified 6X His-tagged recombinant RAN. Purified RAN protein was pulled down by biotin-labeled LINC00858 but not LINC00858 antisense RNA. F Western blotting detection of RAN binding to LINC00858 after FLAG-MCP-MS2 pull-down assays. G, H RIP assays indicated that LINC00858 was precipitated with RAN in whole cell lysates of HGC27 and MKN74 cells (G), and the amount was measured by qPCR (H) and electrophoresis detection (G). I Western blotting of RAN expression in GES1, HGC27, MKN74, SGC7901, BGC823, MGC803, AGS, NUGC4 and SNU216 cells (left panel). Correlations between the mRNA expression of LINC00858 and the protein level of RAN in GC cells (right panel). J Representative images of immunohistochemical analysis of RAN in LINC00858 low- and high-expressing samples (left panel). Associations between LINC00858 expression and protein level of RAN in GC tissues (right panel). The P values in H were calculated using two-sided unpaired Student’s t test, while that in I (right) and J (right) was calculated using Pearson’s correlation analysis and chi-square test. *P < 0.05