Journal: The Journal of Biological Chemistry

Article Title: A Disintegrin and Metalloprotease (ADAM) 10 and ADAM17 Are Major Sheddases of T Cell Immunoglobulin and Mucin Domain 3 (Tim-3) *

doi: 10.1074/jbc.M113.488478

Figure Lengend Snippet: Tim-3 proteolysis is regulated by calmodulin. HEK293 cells were transfected with the expression plasmids for AP-Tim-3, AP-Tim-3 deletion mutant (AP-Tim-3ΔS2), AP-Tim-3 lacking the intracellular domain (ΔICD) or AP-Tim-3 without the intracellular domain with the intracellular lysine at position 226 (ΔICD+1) fusion proteins and treated for 1 h with the calmodulin antagonist W7 (100 μm). The transfected HEK293 cells were incubated in the absence or presence of GI or GW (3 μm, 30 min prior to stimulation) as indicated. The AP activity was calculated as the ratio of alkaline phosphatase activity in the lysate and the conditioned medium (A, C, E, and F). B, D, and G, membrane-bound AP-Tim-3 in cell lysates and soluble Tim-3 in the conditioned medium were subsequently visualized by Western blotting using the anti-hTim-3 IgV-domain mAb (description under “Experimental Procedures”). H, HEK293 cells were transfected with expression plasmid encoding Tim-3 or Tim-3 deletion mutants Tim-3ΔS2 or Tim-3ΔICD without alkaline phosphatase at the N terminus and treated for 1 h with W7 (100 μm). The transfected HEK293 cells were incubated in the absence or presence of GI or GW (3 μm, 30 min prior to stimulation) as indicated. Membrane-bound Tim-3 in the cell lysates and soluble Tim-3 in the conditioned medium were subsequently visualized by Western blotting using anti-hTim-3 IgV-domain mAbs (description under “Experimental Procedures”). The values are from one representative experiment (n = 3). n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t tests). I, HEK293 cells were transfected with expression plasmids for AP-Tim-3, AP-Tim-3 deletion mutant (AP-Tim-3ΔS2), AP-Tim-3 lacking the intracellular domain (ΔICD), or AP-Tim-3 without the intracellular domain with the intracellular lysine at position 226 (ΔICD+1) fusion proteins. Transfected HEK293 cells were lysed and incubated with calmodulin-Sepharose 4B (CaM-Sepharose) or an equivalent amount of Sepharose 4B (Sepharose). Proteins precipitated by immobilized calmodulin were separated on a 15% acrylamide gel, and probed for the presence of Tim-3 by immunoblotting with anti-hTim-3 IgV-domain mAb (description under “Experimental Procedures”). I, HEK293 cells were transfected with the expression plasmids for AP-Tim-3, AP-Tim-3 deletion mutant (AP-Tim-3ΔS2), AP-Tim-3 lacking the intracellular domain (ΔICD), or AP-Tim-3 without the intracellular domain with the intracellular lysine at position 226 (ΔICD+1) fusion proteins. Transfected HEK293 cells were lysed and incubated with calmodulin-Sepharose 4B (CaM-Sepharose) or an equivalent amount of Sepharose 4B (Sepharose). Proteins precipitated by immobilized calmodulin were separated on a 15% acrylamide gel, and probed for the presence of Tim-3 by immunoblotting with anti-hTim-3 IgV-domain mAb (description under “Experimental Procedures”).

Article Snippet: Lysates were incubated with 100 μl of calmodulin-Sepharose 4B or Sepharose 4B (GE Healthcare) in calmodulin buffer (20 m m HEPES, 200 m m KCl, pH 7.4) overnight at 4 °C.

Techniques: Transfection, Expressing, Mutagenesis, Incubation, Activity Assay, Western Blot, Plasmid Preparation, Acrylamide Gel Assay