T cell chemotaxis assays

To quantify if the gp63 D463N mutation reduced T cell migration, we incubated 25 ng/mL of human recombinant CXCL10 with either gp63^PYSDGS, gp63^PYSNGS, or gp63^GYSNGA for 20 hours at 37°C. The CXCL10 and gp63 reaction mixture was added to the basal chamber of a transwell insert (VWR, USA, 10769–236). 250,000 Jurkat T cells engineered to overexpress CXCR3 [23] were added to the apical chamber of the transwell insert. After 2 hours, the cells migrated into the basal chamber were stained using the 7AAD viability dye and quantified using a Guava EasyCyte HT flow cytometer (Millipore, USA).