4.4. Western Blot Analysis

Western blots were performed as previously described [15]. Briefly, total cellular protein (20 µg) was heated to 95 °C and electrophoresed on 4–15% SDS polyacrylamide gels (BioRad, NSW, Australia). Immunoblotting was performed using a semi-dry electrophoresis blotting system (BioRad, Trans-Blot^® Turbo™ System). The membranes were blocked for 2 h in TBST (50 mmol/L Tris-Cl, pH 7.6, 150 mmol/L NaCl and 0.1% Tween 20) containing casein and incubated with the appropriate primary antibody overnight at 4 °C. Primary antibodies used were phosphorylated Akt (1:5000; Cell Signalling, Catalogue number 4058), phosphorylated CREB (1:2000; Cell Signalling, Catalogue number 9198), and phosphorylated ERK (1:2500; Cell Signalling, Catalogue Number 8544). Membranes were washed four times with TBST and HRP-linked secondary Anti-Rabbit antibodies (Cell Signalling, Catalogue Number 7074) were incubated with the membranes for 1 h at room temperature. Membranes were then stripped with Restore Plus Western Blot Stripping Buffer (Thermo Fisher) and probed again for the related housekeeping gene (see Results Figure 3, Figure 4 and Figure 5). All phosphor-immunoreactive species, Akt, CREB, and ERK were normalised against total Akt (1:5000, Cell Signalling, Catalogue Number 9272), total CREB (1:5000, Cell Signalling, Catalogue Number 9197), and ERK (1:5000, Cell Signalling, Catalogue Number 9102). Proteins were visualised with Super Signal West Femto Kit (Thermo Fisher) and imaged using an Odyssey infrared imaging system (LiCor, Millennium Science, Victoria, Australia). Relative band density was quantified using Image J software (https://imagej.nih.gov/ij/). Representative immunoblots are provided from three independent Western blots.