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98 64 50 36. Non-red. Red. Mw in kD. Purified Fc-GITR-L. Fig. S1 Production of Fc-GITR-L fusion protein. S1A Diagram of Fc-GITR-L fusion protein. Human IgG1 Fc Fragment. Mouse GITR Ligand. Transmemberance and intracellular domain. Ig light chain leader sequence.
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98 64 50 36 Non-red. Red. Mw in kD Purified Fc-GITR-L Fig. S1 Production of Fc-GITR-L fusion protein S1A Diagram of Fc-GITR-L fusion protein. Human IgG1 Fc Fragment Mouse GITR Ligand Transmemberance and intracellular domain Ig light chain leader sequence Extracellular domain signal peptide Fc fragment S1B Analysis molecular weight and purity of Fc-GITR-L by SDS-PAGE.
CD4+ T Cells Resting Activated GITR+/+ GITR Fc-GITR-L Fig. S2 The Fc-GITR-L fusion protein binds to its receptor GITR. S2A Fc-GITR-L binds to 293F cells transfected with GITR. GITR Fc-GITR-L S2B Fc-GITR-L binds to GITR in both resting and activated CD4+ T cells.
CD4+CD25– CD4+CD25+ Fc-GITR-L GITR S2C Fc-GITR-L binds to CD4+CD25+ Treg and CD4+CD25 Teff cells.
Fig. S3 Fc-GITR-L induces in vivo proliferation of CD4+FoxP3+ but not CD4+FoxP3 cells S3A Administrating Fc-GITR-L for 2 weeks induces proliferation of Treg but not Teff cells. 2 weeks ns 500 400 ns 300 Cell number(x106) 200 ns 100 0 ns 50 40 ns 30 CD4+(%) ns 20 10 0 IgG IgG IgG Fc-GITR-L Fc-GITR-L Fc-GITR-L Thymus Spleen MLN
S3B Administrating Fc-GITR-L for 4 weeks induces proliferation of Treg but not Teff cells. 4 weeks 500 ns 400 ns 300 Cell number(x106) 200 ns 100 0 ns 50 40 ns 30 CD4+(%) ns 20 10 0 IgG IgG IgG Fc-GITR-L Fc-GITR-L Fc-GITR-L Thymus MLN Spleen
Fig. S4 Fc-GITR-L induces in vivo proliferation of CD4+FoxP3+ cells in both C57BL/6 and BALB/c mice strain S4A Administrating Fc-GITR-L induces proliferation of Treg cells in C57BL/6 mice. *** 20 16 12 8 4 0 C57BL/6 FoxP3+/CD4+(%) IgG Fc-GITR-L S4B Administrating Fc-GITR-L induces proliferation of Treg cells in BALB/c mice. ** 12 8 4 0 BALB/c FoxP3+/CD4+(%) IgG FcGITRL