Fig. 8: S. suis STK phosphorylates SIRT1 to degrade EphA2 for disruption of blood-brain barrier.

A Co-IP analysis of SIRT1-DCAF15 interaction in hCMEC/D3 cells infected with WT SC19, Δstk or CΔstk. B Co-IP analysis of SIRT1-DCAF15 interaction in HEK293T cells co-transfected with Flag-DCAF15, HA-SIRT1 and Myc-STK. C, D Co-IP analysis of STK-SIRT1 interaction in HEK293T cells co-transfected with Myc-STK and HA-SIRT1. E STK-SIRT1 interaction via GST affinity-isolation assay. F Phosphorylation of SIRT1 induced by STK in vitro. G Identification of phosphorylated sites of SIRT1 using mass spectrometry. H Sequence alignment of the conserved Ser48 containing region in SIRT1 orthologs of different species. I Phosphorylation level of SIRT1 in HEK293T cells co-transfected with Myc-STK, HA-SIRT1 or HA-SIRT1-S48A. J Ubiquitination of EphA2 in HEK293T cells co-transfected with HA-Ub, Myc-EphA2, Flag-DCAF15, His-STK, HA-SIRT1-WT or HA-SIRT1-S48A. K Western blot analysis of ZO-1 expression in SC19-infected hCMEC/D3 cells transfected with HA-SIRT1-WT or HA-SIRT1-S48A. Data are representative of three independent experiments with triplicate samples per group. All data are presented as mean ± SD. P ≤ 0.05 was considered as statistical significance.