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Fig. 5 | Journal of Hematology & Oncology

Fig. 5

From: CircRNA-CREIT inhibits stress granule assembly and overcomes doxorubicin resistance in TNBC by destabilizing PKR

Fig. 5

CircRNA-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 0 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-293 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-293 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-293 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 result

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