Figure 5.

Sensitivity of p53 in HCMV‐infected cell lysates to cell‐free cleavage by purified µ‐ or m‐calpain and generation of p53(ΔCp44) by purified m‐calpain. HCMV‐infected cells were treated with or without 100 µM E64d 6 hours before harvest to help preserve calpain‐sensitive p53. Nuclear protein (160 µg) was isolated at the indicated time post infection (PI) and incubated with 0.08 units or less of purified µ‐calpain or m‐calpain for 10 minutes at 30°C. In one of the two control samples in each cleavage assay, calpain was replaced with EDTA, which inactivates endogenous Ca²⁺‐dependent proteases (lane 1). Protein extracts (80 µg) were then analyzed by immunoblot with DO‐1 and reprobed with actin antibody. The results are representative of at least two independent biological replicates, each one in two technical replicates. Arrowhead: p53(ΔCp44). kDa sizes: the relevant molecular weight markers