Research Paper Volume 12, Issue 12 pp 11685—11697

Figure 4. LncRNA-KCNQ1OT1 directly binds and stabilizes HK2. (A) Proteins retracted from the KCNQ1OT1 pull-down assay are dissected by SDS-PAGE for mass spectrometry assay. (B) Western blotting analysis of KCNQ1OT1-interacting proteins that are pulled down in the RNA pull-down assays using biotinylated KCNQ1OT1. (C) RNA immunoprecipitation (RIP) assay results show that the KCNQ1OT1 RNA is pulled down with the anti-HK2 antibody in HCT116 cell. (D) QRT-PCR analysis of KCNQ1OT1 in the total RNA that is pulled down using the anti-HK2 antibody in the RIP assay, thereby confirming the direct interaction between HK2 protein and KCNQ1OT1. (E) Representative western blot images show HK2 protein levels obtained from incubating total protein extracts from HCT116 cells with biotinylated RNAs containing different regions of KCNQ1OT1 and the negative control RNA followed by the RNA pull down assay. The blots are probed using the anti-HK2 antibody. (F) Representative western blot images show HK2 protein levels in sh-NC- and sh-KCNQ1OT1-transfected HCT116 cells. (G) QRT-PCR results show HK2 mRNA levels in sh-NC- and sh-KCNQ1OT1-transfected HCT116 cells. (H) Representative western blot assay results show HK2 protein levels in sh-NC- and sh-KCNQ1OT1-transfected HCT116 cells treated with 100 μg/mL cycloheximide for 0, 4, and 5 h before harvesting the cells for analysis. Untreated cells are used as controls. (I) Histogram plot shows HK2 protein levels in sh-NC- and sh-KCNQ1OT1-transfected HCT116 cells treated with 100 μg/mL cycloheximide for 0, 4, and 8 h. (J) Representative western blot images show HK2 protein levels in sh-NC- and sh-KCNQ1OT1-transfected HCT116 cells treated with 10mM MG132 for 4h. (K) Representative western blot images shows ubiquitinated HK2 protein levels in sh-NC- and sh-KCNQ1OT1-transfected HCT116 cells. Note: **p < 0.01; NS, not significant