Fig. 3

Caspy2 directly binds to LPS to induce its oligomerization. a Streptavidin pulldown assays of the binding of biotin-conjugated LPS and Pam3CSK4 to purified HA-tagged caspy2, caspy2 (C296A), caspy2 (?PYD), caspy2 PYD, caspy and caspy PYD in transfected HEK293T cells. Shown are anti-HA immunoblots of pulled down proteins and total lysates (input). b Purified HA-tagged caspy2 and caspy2 PYD in transfected HEK293T cells were incubated with LPS or Pam3CSK4. The samples were analyzed by the pore-limited native gel electrophoresis. c Wild-type and caspy2-KO ZF4 cells were infected with wild-type (EIB202) or 0909I E. piscicida (with/without 10?mM glycine) for 2?h at a multiplicity of infection (MOI) of 50. Confocal laser scanning microscopic analysis of nuclei (2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI), blue) and caspy2 foci (green, white arrowheads). Direct interference contrast (DIC)/phase images are shown. Scale bar, 10?µm. Statistics of the percentages of cells showing signals for caspy2 foci are listed below. (Approximately 200 cells were counted in each sample. Mean?±?SD of triplicate samples.) d Wild-type and caspy2-KO ZF4 cells were infected with wild-type (EIB202) or 0909I E. piscicida (with/without 10?mM glycine) for 2?h at an MOI of 50, or left untreated (Mock). The samples were analyzed by the pore-limited native gel electrophoresis and immunoblotting (upper panel). Cell lysates and DSS cross-linked pellets from wild-type and caspy2-KO ZF4 cells treated as indicated were analyzed by immunoblotting for caspy2 oligomerization (lower panel). WB western blot. Results are representative of at least three independent experiments