Figure 2.

In vitro mechanism of action studies and the synthesis of ⁶⁴Cu-GRIP B. (A) Mean fluorescence intensity data showing the extent of cell labeling by 5FAM-GRIP B in the presence or absence of GZMB after incubating at 37 °C for 30 min. The data were collected using MC38 cells in triplicate. *P < 0.01. (B) A bar graph representing the extent of red blood cell lysis due to treatment with vehicle (0.1% dimethyl sulfoxide (DMSO)), the full-length GRIP B pro-peptide, and the proteolytically activated truncated peptide. Triton-X is included as a positive control. (C) An HPLC trace showing the overlay of the radioactive trace (blue) with the UV trace of the DOTA-GRIP B precursor. The trace was collected 30 min after the start of the reaction. (D) A radioactive HPLC trace showing the conversion of ⁶⁴Cu-GRIP B to one major product after a 30 min incubation with 10 nM recombinant human GZMB.