Fig. 5From: CircRNA-CREIT inhibits stress granule assembly and overcomes doxorubicin resistance in TNBC by destabilizing PKRCircRNA-CREIT enhanced the binding of HACE1 and PKR proteins. A Western blotting for PKR protein levels after HACE1 overexpression or knockdown in TNBC cells. B, C TNBC cells stably transfected with sh-HACE1 plasmids or empty vectors were treated with CHX for the indicated times. Western blotting (B) and statistical analysis (C) of PKR protein levels are shown, with the level at 0Â h as a control. D The impact of HACE1 on the PKR ubiquitination level was verified by co-IP assays and subsequent Western blotting. IB: immunoblot. E K48-linked ubiquitination of PKR was enhanced by HACE1 overexpression. IB: immunoblot. HA-Ub-K48only: the cells were transfected with plasmids expressing HA-tagged ubiquitin with all lysines mutated except K48. HA-Ub-K63only: the cells were transfected with plasmids expressing HA-tagged ubiquitin with all lysines mutated except K63. F HEK-293Â T cells transfected with HACE1-Flag or PKR-Flag were lysed, immunoprecipitated with anti-Flag and then subjected to Western blotting assays using anti-PKR or anti-HACE1, respectively. G, H HEK-293Â T cells co-transfected with PKR-Flag and HACE1-Myc or the corresponding empty vectors were lysed, immunoprecipitated with anti-MYC (G) or anti-Flag (H), and subjected to Western blotting analysis. I HEK-293Â T cells were cotransfected with PKR-Flag, HACE1-Myc and circRNA-CREIT overexpression vectors or the corresponding empty plasmids. Co-IP assays validated that circRNA-CREIT increased the PKR-Flag level precipitated by HACE1-Myc. Three independent experiments were conducted for each resultBack to article page