Fig. 6: SYVN1 ubiquitinates GSDMD with K27-linked polyubiquitin chains.
From: E3 ubiquitin ligase SYVN1 is a key positive regulator for GSDMD-mediated pyroptosis
A Immunoprecipitation analysis of HEK293T cells co-expressing Flag-GSDMD and HA-Ub together with Myc-SYVN1 or Myc-SYVN1-C329S. Anti-Flag immunoprecipitates were analyzed using immunoblotting with indicated antibodies. The expression levels of the transfected proteins were analyzed using immunoblotting with indicated antibodies. B Immunoprecipitation analysis of HEK293T cells co-expressing Flag-GSDMD and Myc-SYVN1 together with HA-Ub (K48, K63, K48R, or K63R) as indicated. Immunoblotting analysis was performed using anti-Myc or anti-Flag antibodies. Expression of the transfected proteins was analyzed using immunoblotting, using corresponding antibodies. C Immunoprecipitation analysis of HEK293T cells expressing Flag-GSDMD and Myc-SYVN1 together with HA-Ub (K6, K11, K27, K29, K33, K48, or K63 only) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibodies. Expression of the transfected proteins was analyzed using immunoblotting, using corresponding antibodies. D Immunoprecipitation analysis of HEK293T cells expressing Flag-GSDMD and Myc-SYVN1 together with HA-Ub (K27 or K27R) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with indicated antibodies. Expression of the transfected proteins was analyzed using immunoblotting, using corresponding antibodies. E LDH release assay of HEK293T cells transfected with caspase-1/4, GSDMD and Ub-K27 or control vector. F LDH release assay of HEK293T cells transfected with hGSDMD-p30 and Ub-K27 or control vector. All data were representative of three independent experiments.
