Research Paper Volume 12, Issue 20 pp 20396—20412

Figure 4. FAM83C-AS1 stabilized EZH2 protein by binding to ZRANB1 in CRC. (A) EZH2 protein expression was analyzed using western blotting analysis in different groups. (B) After treatment with CHX (50 μg mL^-1), EZH2 protein stability was evaluated in RKO and SW620 cells transfected with sh-FAM83C-AS1#1 or sh-NC. (C, D) The binding capacity between FAM83C-AS1 and ZRANB1 was measured using RNA pull-down assay. (E) RIP assays further proved that FAM83C-AS1 could bind to ZRANB1 in RKO and SW620 cells. (F) mRNA and protein expression of ZRANB1 in RKO and SW620 cells transfected with sh-FAM83C-AS1#1 or sh-NC were examined using RT-qPCR and western blotting analysis. (G) The efficiency of ZRANB1 knockdown or overexpression was assessed using RT-qPCR analysis. (H) FAM83C-AS1 expression in transfected cells was detected by RT-qPCR. (I) CoIP assay was adopted for analyzing the effect of FAM83C-AS1 depletion on the binding of ZRANB1 to EZH2. (J) The relationship between EZH2 ubiquitination and FAM83C-AS1 or ZRANB1 was analyzed in different groups. ^**P < 0.01. n.s.: no significance.