Figure 6. Impact of carbamylation on proteasome proteolytic activities and on the ubiquitination process. (a) Evaluation of proteasome proteolytic activity after incubation of cells with urea or cyanate: confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions, open bars) or with 20 mmol/L urea (grey bars) or 0.5 mmol/L cyanate (black bars). Chymotrypsin-like, caspase-like and trypsin-like activities have been measured in cell extracts using the corresponding Proteasome-Glo™ assays. The data are presented as means ± SEM (n=6) and compared using the Mann-Whitney U test (ns: non significant, **: p<0.01). (b) Ubiquitination level of intracellular proteins after incubation of cells with urea or cyanate: confluent fibroblasts were incubated for 4 weeks at 37°C with DMEM + 0.5% (v/v) FBS without (control conditions) or with 20 mmol/L urea or 0.5 mmol/L cyanate, and cell extracts were prepared and submitted to western-blot analysis using an anti-ubiquitin antibody. (c) Anti-HCit and anti-ubiquitin immunolabellings were performed using fibroblasts previously seeded (10,000 cells/well) in chambered coverglass system and incubated for 2 days with DMEM containing 0.5% (v/v) FBS and 5 mmol/L cyanate. At the end of incubation, cells were fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.25% (v/v) Triton X-100 before immunolabelling of proteins using both anti-HCit and anti-ubiquitin antibodies. Colocalization points between HCit and ubiquitin labelling were identified using ImageJ software.