The P5 preferences of caspases 2, 3, and 8 determined using the Ac-P5-Mix-Glu-Mix-Asp-ACC library were described in our previous manuscript (16). Herein, we used the same approach to screen P5 preferences of other apoptotic caspases, including caspases 6, 7, 9, and 10. Each caspase was tested in the appropriate assay buffer (the buffer for caspases 9 and 10 was supplemented with sodium citrate). The P5 library was used at 100 μM, and the caspase concentrations were as follows: caspase 6, 50 nM; caspase 7, 20 nM; caspase 9, 50 nM; and caspase 10, 20 nM. All screenings were performed on 96-well plates in a 100 μl volume. The total assay time was 30 min, but only the linear portion of the fluorescence progress curve was taken for the analysis. For each caspase, the hydrolysis rate of the control tetrapeptide substrate (Ac-Mix-Glu-Mix-Asp-ACC) lacking the P5 position was set as 100% (or 1.0), and the P5 substrates were adjusted accordingly.