Fig. 2

LINRIS was associated with IGF2BP2 in CRC. a IGF2BP2 was pulled down by biotin-labeled sense LINRIS (S) but not LINRIS anti-sense (AS) RNA in the indicated cells. b RIP assays were applied using anti-IGF2BP2 antibodies with extractions from HCT116 cells. Relative enrichment (mean ± SD) represents the RNA levels associated with the indicated protein relative to an input control from three independent experiments after immunoprecipitation with the anti-IGF2BP2 antibody compared with that with the IgG antibody. MYC mRNA was uesd as the positive control and GAPDH mRNA was used as the negative control. c Expression vectors for FLAG-tagged MCP and MS2-tagged LINRIS were transfected into CRC cells to establish the FLAG-MCP-MS2 system. And IGF2BP2 was then pulled down using the anti-FLAG® M2 affinity gel followed by the Western blot analysis. d Western blot detection of IGF2BP2 binding to LINRIS after FLAG-MCP-MS2 pull-down assays. e In vitro-synthesized full-length (FL), N-terminal (NT) and C-terminal (CT) fragments of LINRIS were incubated with protein lysates from HCT116 cells. RNA pull-down and Western blotting assays were then performed. The data shown represent three independent experiments. f Western blot analysis shows the levels of IGF2BP2 in 11 CRC cell lines with GAPDH as the loading control. g IGF2BP2 expression was positively correlated with LINRIS expression in CRC cells. The r values and P values are from Pearson’s correlation analysis. h Western blot analysis shows the expression of IGF2BP2 with or without knockdown of LINRIS in the indicated cells. i CRC cells transfected with shRNAs specific for LINRIS or a scrambled control. Cell lysates were immunoprecipitated with either an antibody against IGF2BP2 or an IgG control and then analyzed by immunoblotting with a ubiquitin (Ub)-specific antibody