Figure 5.

⁴²RRR⁴⁴ patch is required for general protein hydrolysis in cell lysates. A–C, lysates generated from SK-N-AS neuroblastoma cells were incubated with 100 nm of either WT caspase-6, R42–44A, or R44K to assess hydrolysis over time by Western blotting of substrate proteins. A, PARP; B, lamin A/C; or C, DJ-1 normalized to loading control β-actin. D–F, quantification of the data from WT (black), R42–44A (white), or R44K (gray) normalized to the loading control for protein substrates PARP (D), lamin A/C (E), or DJ-1 (F) demonstrates that the hydrolysis of several biologically relevant substrates of caspase-6 are impacted by the removal of the ⁴²RRR⁴⁴ exosite. Changes in substrate processing of multiple proteins suggest they are engaging caspase-6 through a similar mechanism. Experiments were performed in triplicate. *, ** denotes 90 or 95% confidence interval by unpaired two-tailed t test of the variants individually compared with the WT.