GrK and GrKSA were produced as described previously (25). Briefly, Human GrK and its catalytically inactive mutant GrKSA, in which a serine has been replaced with Ala, were produced in Pichia pastoris and purified using cation-exchange chromatography. After dialysis granzymes were further purified (to >98% homogeneity) by affinity-chromatography using Protein A/G Ultralink beads (Thermo Scientific) and the catalytic activity was confirmed using the synthetic chromogenic substrate Ac-Lys-pNA (Bachem) and cleavage of macromolecular substrates. The GrK concentrations used in our experiments are based on previous work that studied extracellular effects of granzymes (23, 24).

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