Figure 3.

SCARA1 recognizes dead cells through cellular spectrin. A, CC–CL fragment of mSCARA1 does not bind to the ActD-treated Jurkat cells. Mock represents untreated viable cells. B, CL–SRCR fragment of mSCARA1 binds to the ActD-treated Jurkat cells in the presence of Ca²⁺. GFP is applied as a control. C, trimeric 5J0J–SRCR binds to the ActD-treated Jurkat cells in the presence of Ca²⁺. D, dot-blot assays show that the ectodomain and the CL–SRCR fragment of mSCARA1 bind to the Jurkat cell lysates in the presence of Ca²⁺, whereas the CC–CL fragment has no detectable binding to the cell lysates. Serially-diluted cell lysates were spotted on the nitrocellulose membranes. E, mouse SCARA1 shows no binding to the lipids on the strip. Jurkat cell lysates were spotted as a positive control. F, confocal image of the permeabilized HEK293 cells stained by the CL–SRCR fragment of hSCARA1 and DAPI in the presence of Ca²⁺ (scale bar, 25 μm). G, confocal image of the permeabilized NIH 3T3 cells stained by the CL–SRCR fragment of mSCARA1 and DAPI in the presence of Ca²⁺ (scale bar, 25 μm). H, monomeric SRCR domain of mSCARA1 shows weaker binding to the ActD-treated Jurkat cells than the trimeric mSCARA1 ectodomain. GFP is applied as a control. I, monomeric SRCR domain of hSCARA1 shows weaker binding to the ActD-treated Jurkat cells than the trimeric hSCARA1 ectodomain. J, binding of mSCARA1 to dead cells is abolished by the protease K treatment. K, dot-blot assays show that spectrin can be pulled down by GFP–hCL–SRCR fragment from the Jurkat cell lysates. The cell lysates were incubated with the Ni-NTA beads bound with the purified GFP–hCL–SRCR fragments in the presence of Ca²⁺ or EDTA, and the pulldown products were spotted onto nitrocellulose membranes and detected by anti-spectrin or anti-GFP antibodies. The pulldown products from the empty Ni-NTA beads as well as the GFP–hCL–SRCR fragment alone were also spotted as controls. L, staining of viable (left) and the ActD-treated (middle) Jurkat cells by annexin V-APC, PI, and anti-spectrin antibodies. The binding of anti-spectrin antibodies to the gated subsets of viable and the ActD-treated Jurkat cells is shown on the right. Mock represents untreated viable cells.