Fig. 5: LegK3 targets multiple caspases.
From: Phosphorylation of caspases by a bacterial kinase inhibits host programmed cell death
A His6-Caspase-7*, His6-Caspase-8* or His6-Caspase−9* was purified from E. coli strains co-expressing Flag-LegK3 or Flag-LegK3D/A. After separation by SDS-PAGE, phosphorylation of these caspases was either assessed by Pro-Q diamond phospho-protein staining or analyzed by immunoblotting by phospho-(Ser/Thr)-specific antibodies. Caspase-7*, Caspase-8* and Caspase-9* represent for Caspase-7C186A, Caspase-8C360A, and Caspase-9C287A, respectively. B Extracted ion chromatograms of the Ser199-phosphorylated peptide (GTELDDGIQADpSGPINDTDANPR) and a control peptide (SSFVPSLFSK) from Caspase-7 used in (A). C Extracted ion chromatograms of the Thr102-phosphorylated peptide (LSKPpTLENLTPVVLRPEIR) and a control peptide (TGSNIDCEKLR) from Caspase-9 used in (A). D, E Caspase-7*S199A and Caspase-9*T102A purified from E. coli co-expressing wild-type LegK3 or LegK3D/A were probed by Pro-Q diamond phospho-protein staining or by immunoblotting using anti-Phospho-(Ser/Thr) antibodies. Caspase-7* and Caspase-9* represent for Caspase-7C186A and Caspase−9C287A, respectively. F, G HA-Caspase-7S199A or HA-Caspase−9T102A was co-expressed with GFP-LegK3 or GFP-LegK3D/A in HEK293T cells by transfection. After immunoprecipitation, phosphorylation of Caspase-7/−9 was evaluated by immunoblotting with anti-Phospho-(Ser/Thr) antibodies. Data in A, D, E, F, and G are representative from three independent experiments. Source data are provided as a Source Data file.
