Quantity one 4.2.1 image analysis software

Tissue lysates and Western blotting were prepared as previously described [12] (link). Twenty micrograms of protein was separated on polyacrylamide gels (Bio-Rad Laboratories; Gladesville, NSW, Australia) and transferred to PVDF. Protein expression was identified by comparison with the mobility of protein standard. Blots were incubated with rabbit polyclonal phosphorylated (Ser21/9) GSK3α/β (#9331; Cell Signalling, Beverly, MA, USA) or rabbit monoclonal total GSK3β (#9315, Cell Signalling, Beverly, MA, USA) diluted 1/1000 in blocking buffer (3% BSA in TBS with 0.05% Tween-20) for 16 h at 4°C. Membranes were viewed and analysed using the Chemi-Doc system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Semi-quantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad Laboratories; Gladesville, NSW, Australia). The levels of phosphorylated GSK3α/β were normalised to the levels of total GSK3β and fold change was calculated relative to the NGT group.

Western blotting was used to determine the effect of selenium on phosphorylation (i.e., activation) of the MAPK signalling pathway protein ERK. Western blotting was performed as previously described [52 (link)]. Mouse monoclonal pERK (sc-7383; Santa Cruz Biotechnology) and rabbit polyclonal ERK (sc-93; Santa Cruz Biotechnology, Dalla, Texas, USA) were used at 0.2 μg/mL. Membranes were viewed and analysed using the ChemiDoc MP system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Semi-quantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad Laboratories, Hercules, CA, USA).

Protein extraction and Western blotting were performed as previously described [39 (link)]. Twenty micrograms of protein were separated into 10% polyacrylamide gels and transferred to nitrocellulose. Blots were incubated in either 1 μg/ml mouse monoclonal anti-Ras (clone RAS10; Merck Millipore; Billerica, MA, USA), 1 μg/ml mouse monoclonal antiphosphorylated (Tyr204) ERK (p-ERK) (sc-7383; Santa Cruz Biotechnology; Santa Cruz, CA), or 1 μg/ml total ERK1/2 (sc-93; Santa Cruz Biotechnology; Santa Cruz, CA, USA) prepared in blocking buffer (5% skim milk in TBS with 0.05% Tween-20) for 16 h at 4°C. Membranes were viewed and analysed using the ChemiDoc XRS system (Bio-Rad Laboratories; Gladesville, NSW, Australia). Semiquantitative analysis of the relative density of the bands in Western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad Laboratories, Hercules, CA, USA). The levels of Ras were normalised to the levels of β-actin (Sigma, St. Louis, MO, USA). The levels of p-ERK were normalised to the levels of total ERK1/2.