Fig 1: CBP mediates cGAS crotonylation.A, IB analysis was performed on WCLs and anti-GFP IPs of HEK293T cells transfected with the indicated constructs. B, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with increasing amounts of HA-CBP plasmid (1 μg, 2 μg, or 4 μg). C, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with the indicated siRNA and plasmids. D and E, Co-IP of endogenous cGAS with CBP from HeLa cells. CBP and cGAS were immunoprecipitated using anti-cGAS and anti-CBP antibodies, respectively. F, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with plasmids encoding full-length Flag-cGAS and Flag-cGAS truncation mutants, and interactions with CBP were detected. CBP, CREB-binding protein; cGAS, cyclic GMP-AMP synthase; IB, immunoblotting; IP, immunoprecipitate; WCLs, whole-cell lysates.
Fig 2: SIRT3 mediates cGAS decrotonylation.A, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with the Flag-cGAS plasmid and pretreated with 1 μM TSA or 10 mM NAM for 10 h to observe the effects on cGAS crotonylation levels. B, IB analysis was performed on WCLs and anti-GFP IPs of HeLa cells transfected with the indicated constructs. C, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with increasing amounts of Myc-SIRT3 plasmid (1 μg, 2 μg, or 4 μg). D, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with the indicated siRNAs and plasmids. E, Co-IP of endogenous SIRT3 with Flag-cGAS from HeLa cells at the indicated times post-IR (8 Gy γ-ray). F, in vitro cell-free reaction assay of cGAS crotonylation and decrotonylation by CBP and SIRT3, respectively. Workflow of the in vitro assay (upper panel). The samples were analyzed by IB. cGAS, cyclic GMP-AMP synthase; IB, immunoblotting; IP, immunoprecipitate; NAM, nicotinamide; TSA, trichostatin A; WCLs, whole-cell lysates.
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