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 (ΔPYD), 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