Fig. 9

Pro-caspase-4 interacts with pro-caspase-1 in E. histolytica-stimulated THP-1 macrophages. a A schematic workflow of cell preparations to quantify caspase-1 and caspase-4 interaction by pull-down and western blot. Cells were incubated with Eh, treated with formaldehyde, lysed and cross-linked complexes (black triangles with oval shapes) were pulled-down (PD) with antibodies (Y-shaped) and protein A/G plus agarose beads (gray circles). b–e THP-1 macrophages or COS-7 cells were incubated with Eh for 20 min and 0.8% formaldehyde treated for 7 min at room temperature. Cells were subjected to pull-down (PD) with caspase-4 M029-3 or caspase-1 sc-56036 antibody at 1.5 μg and immunoblotted for caspase-4 or caspase-1. f, g COS-7 cells were transfected with caspase-1 (pC-1, 60 ng), caspase-4 (pC-4, 500 ng), or IL-1β (pIL-1β, 1.5 μg) plasmids, or GFP (pGFP, 1 μg) as a control prior to incubating with Eh (2:1). Cells were subjected to pull-down with caspase-4 M029-3 or caspase-1 sc-56036 antibody at 1.5 μg and immunoblotted for caspase-4 or caspase-1. Corresponding mouse (mIgG) controls were used. Lysates were loaded onto SDS-PAGE gel and immunoblotted with the indicated antibodies. Western blots are representative of three independent experiments