Fig. 4: Phosphorylation of Caspase-3 at Ser29 by LegK3 inhibits its cleavage by initiator caspases.
From: Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death
A–D. His6-Caspase-3*, phosphorylated His6-Caspase-3* or His6-Caspase-3*S29A was incubated with His6-Caspase-9140-C (A, B) or His6-Caspase−8 (C, D) for the indicated durations. Cleavage of Caspase-3* was visualized by Coomassie brilliant blue staining (CBB) after SDS-PAGE. Quantitation was determined by measuring the density of the bands corresponding to uncleaved Caspase-3* (B, D). Caspase-3* represents for Caspase-3C163A. Caspase-9140-C expressed in E. coli produces the active form of Caspase-9. E 4xFlag-Caspase-3* purified from HEK293T cells co-transfected with GFP-LegK3 or GFP-LegK3D/A was incubated with His6-Caspase-9140-C. Cleavage of Caspase-3 was detected by immunoblotting. Caspase-3* represents for Caspase-3C163A. F–I Phosphomimetic mutants of Caspase-3* purified from E. coli were reacted with recombinant active Caspase-9140-C and the cleavage was evaluated by CBB staining. Quantitation was determined by measuring the density of the bands corresponding to uncleaved Caspase-3* (G and I). Caspase-3* represents for Caspase-3C163A. Data in A, C, E, F, and H are representative from three independent experiments. For B, D, G, and I, data shown are mean ± SD from three replicates. Unpaired two-tailed Student’s t tests were performed. Source data are provided as a Source Data file.
