Chemidoc mptm imaging system

Supernatant was removed from cells following stimulation, and lysates were collected in 30–50 μl lysis buffer (0.125 M Tris pH 6.8, 10% glycerol, 0.02% SDS and 5% DTT) Lysates were subsequently heated to 95 °C for 5 min to denature proteins. SDS–PAGE was used to resolve proteins by molecular weight. Samples were boiled at 95 °C for 5 min before loading into a 5% stacking gel. The percentage resolving gel depended on the molecular weight of the given protein. The Bio-Rad gel running system was used to resolve proteins, and the Bio-Rad wet transfer system was used for the electrophoretic transfer of proteins onto a PVDF membrane. Following transfer, the membrane was incubated in milk powder (5% in TBST) for 1 h and subsequently incubated in primary antibody rolling overnight at 4 °C. Primary antibodies targeting phospho-proteins were diluted in BSA (5% in TBST) as opposed to milk. The membrane was incubated for 1 h with secondary antibody (diluted in 5% milk powder) at room temperature. Before visualization, the membrane was immersed in WesternBright ECL Spray (Advansta). Protein visualization was performed using a ChemiDoc MPTM imaging system (Bio-Rad), and both chemiluminescent and white light images were taken. Images were analysed using Image Lab 6.0.1 (Bio-Rad).