Fig. 5

HOXC-AS3 Interacts with YBX1. a After nuclear and cytosolic separation, RNA expression levels were measured by qRT-PCR. GAPDH was used as a cytosol marker and U6 was used as a nucleus marker. b For the in vitro transcribed, pull-down assays-MS and WB assays showed that desthiobiotinylation-HOXC-AS3 could bind YBX1 in BGC-823 cells. c RIP experiments for YBX1 (two different antibodies from Abcam) were performed and the coprecipitated RNA was subjected to qRT-PCR for HOXC-AS3. The fold enrichment of HOXC-AS3 in RIPs is relative to its matching IgG control RIP. LncRNA MEG3 was a negative control. d Using a series of HOXC-AS3 deletion mapping constructs and RNA pull-down-WB assays, the YBX1-binding activity mapped to nucleotides 115 nt of HOXC-AS3. The profiles are established by RNA pull-down of the BGC-823 extract, and the retrieved proteins are detected by immunoblotting. e The qPCR and western blot assays detected the expression of YBX1 after knockdown of HOXC-AS3. *P < 0.05, n.s., not significant