Figure 4. Resveratrol inhibits the chymotrypsin-like activity of 20S proteasome and resulted in an increased accumulation of protein-ubiquitin conjugates. Δ16HER2 and β-actin (internal control) mRNA levels were measured by semi-quantitative RT-PCR (a) and qRT-PCR (b) analyses in spontaneous mammary tumors from Δ16HER2 mice supplemented or not with resveratrol. (c) The chymotrypsin-like activity of the 20S proteasome was measured in tumor samples, from Δ16HER2 mice treated or not with resveratrol, as described in Materials and Methods, and expressed as fluorimetric units (FU) min-1 mg-1. The significance was determined by unpaired two-tailed student t test, *p < 0.05. (d) Western blot analysis of 20S proteasome subunit content (upper panel) and ubiquitin-protein conjugates and free ubiquitin levels (lower panel) in tumor samples from Δ16HER2 mice treated or not with resveratrol. β-actin was used as loading control. (e) Representative western blot analysis of p53 and β-actin (loading control) in spontaneous mammary tumors from Δ16HER2 mice, supplemented or not with resveratrol, and (f) relative densitometric quantification from three independent experiments. The significance was determined by unpaired two-tailed student t test, *p < 0.05.