Research Paper Volume 11, Issue 11 pp 3624—3638

Proteasome-dependent degradation of intracellular carbamylated proteins

Figure 3. Localization of intracellular carbamylated proteins. Fibroblasts were seeded in chambered coverglass system and incubated for 5 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 carbamylated proteins using an anti-HCit polyclonal antibody (a). Cells were also labelled using ActinRed 555 ReadyProbes® in order to identify actin fibers (b). Colocalization points between HCit and actin labelling were identified using ImageJ software (c). In a second set of experiments, fibroblasts were incubated in the same conditions without (control) or with 5 mmol/L cyanate before preparing total cell extracts which were then used for β-actin immunoprecipitation. The immunoprecipitates were submitted to acid hydrolysis before HCit quantification by LC-MS/MS (d). The data are presented as means ± SEM (n=4) compared using the Mann-Whitney U test (**: p<0.01).