Gs170 calibrated imaging densitometer

Protein expression of proliferating cell nuclear antigen (PCNA) was studied by means of immunoblotting as previously described (Bajt et al. 2000 (link); Maes et al. 2016b (link)). In essence, liver tissue protein lysates (50 μg per lane) were resolved on 4-20% sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Corporation, USA) and blocked overnight at 4°C with 5% milk in TBS/T. After washing with TBS/T, membranes were incubated with a mouse monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, USA), diluted 1/2000 in blocking buffer for 2 hours at room temperature. Membranes were washed and incubated with appropriate secondary horseradish peroxidase-coupled antibody (Santa Cruz Biotechnology, USA) for 1 hour at room temperature. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, USA) according to the manufacturer’s instructions. Densitometric analysis was performed with a GS170 Calibrated Imaging Densitometer (Bio-Rad, USA) using Quantity One 4.0.3. software (Bio-Rad, USA). For semiquantification purposes, PCNA signals were normalized against β-actin signals.

Protein expression of PCNA was studied by immunoblotting as described elsewhere (33 (link)). Protein concentrations were determined by the Bradford procedure (34 (link)) by using a commercial kit (Bio-Rad, USA) with bovine serum albumin as a standard. Proteins were separated on 12% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, USA) under reducing conditions and transferred to nitrocellulose membranes with iBlot^® Transfer Stacks (Life Technologies, USA). Membranes were incubated with a mouse monoclonal anti-PCNA antibody (Santa Cruz, USA) overnight at 4°C and incubated with a secondary horseradish peroxidase-coupled anti-mouse antibody (Dako, USA) at room temperature for 1 hour. Enhanced chemiluminescence was used to visualize proteins (Amersham Biosciences, USA). Densitometric analysis was performed with a GS170 Calibrated Imaging Densitometer (Bio-Rad, USA). For semi-quantification purposes, PCNA signals were normalized against β-actin signals (Sigma, USA) and expressed as relative alterations compared to WT animals.