2.4. Western Immunoblotting

Cells were seeded in 6-well plates at a density of 2.5 × 10⁵/mL in 2 mL supplemented medium and left to adhere for 24 h before the drug of interest was added. After 24 h, medium containing drug was removed and cells washed with 2 mL cold PBS. 40 µL of sodium dodecyl sulfate (SDS) lysis buffer was added to each well and lysates collected. Lysates were heated at 100 °C for 10 min, and then sonicated three times at amplitude 6.0 for 10 s using a MSE Soniprep 150 Plus ultrasonic disintegrator (Henderson Biomedical Ltd., Sydenham, UK). A Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Cramlington, UK) was used to quantify the amount of protein in each lysate. From each assay, 35 µg was then loaded onto a 12-well 4–20% Mini-PROTEAN® TGX™ Gel (Bio-Rad, Hertfordshire, UK), the outside wells loaded with SeeBlue™ Pre-stained Protein Standard (Invitrogen, Cramlington, UK) and gel electrophoresis separation of the proteins performed at 180 V for 45 min. The separated proteins were subsequently transferred to a Hybond-C nitrocellulose membrane by orthogonal electrophoresis at 100 V for 30 min. The membrane was then blocked for 1 h in either 5% milk/TBS/Tween or BSA. The membrane was cut into three and each strip incubated in primary antibody overnight at 4 °C. Membranes were washed in TBS/Tween then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at a 1:1000 dilution for 90 min at room temperature. Membranes were then subject to 4 × 4 min washes in TBS/Tween and imaged using a G:BOX XT4 Chemiluminescence and Fluorescence Imaging System (Syngene, Cambridge, UK), using Clarity Western Enhanced Chemiluminescence (ECL) Substrate (Bio-Rad, Hertfordshire, UK). To remove antibodies, membranes were then incubated in harsh stripping buffer (20% (v/v) SDS, 12% (v/v) 0.5 M Tris HCl pH 6.8, 67% (v/v) ultra-pure water and 1% (v/v) βeta-mercaptoethanol) in a water bath at 56 °C with agitation for 30 min, before being washed twice with TBS/Tween. For re-probing with different antibodies, the membranes were blocked and the same procedure as mentioned above followed. For details of primary antibodies used see Table A1. Secondary antibodies were from Dako (Copenhagen, Denmark): polyclonal Goat Anti-Rabbit (PO448) and polyclonal Goat Anti-Mouse (PO447). Original western blots are included in Figures S1 and S2.