Cellular Thermal Shift Assay (CETSA). Approximately 1.0×10 6 HEK293T cells were seeded into treated 6-well plates before incubating at 37 °C overnight before being treated with the desired amounts of compound in serum supplemented DMEM (5% FBS, 1x Pen/Strep), with DMSO concentrations normalized to 0.1% for all samples. Cells were dosed in 6-well treated plates for 2 h and incubated at 37 °C. Upon completion of the incubation period, cells were pelleted at 300x g and rinsed in PBS before being resuspended in 100 µL PBS. Re-suspended cells were thermally denatured at 48 °C for 3 min in a heat block and subsequently equilibrated at room temperature for a further 3 min. Cells were pelleted at 300x g, PBS decanted, and resuspended in PBS supplemented with 1× cOmplete Mini Protease (Roche) before being lysed over three freeze thaw cycles and centrifuged (15 min at 4 °C, 15,000 X g.). Soluble protein within the supernatant was separated and total protein concentration was determined using the BCA protein assay kit (Pierce). Samples were normalized to the lowest total protein concentration. Normalized samples were mixed with 4× NuPAGE LDS loading buffer (Invitrogen) and 10x reducing agent (Invitrogen), and heated at 95 °C for 5 minutes before separation on 3-8% Tris-Acetate gels. Proteins were transferred to BioRad PVDF membranes for 10 minutes on a BioRad Trans-Blot Turbo. Membranes were dried at ambient temperature for 1 h and cut at 70 kDa. The membranes were blocked in TBS-T containing 5% nonfat dry milk, and subsequently incubated with TBS-T containing primary antibodies at dilutions listed below. Primary antibody solutions were incubated at 4 °C for 18 h, before washing three times with TBS-T. Membranes were then incubated with secondary antibodies for 2 h at room temperature. Membranes were washed five times in TBS-T and treated with SuperSignal West Dura substrates (Thermo) for 1 min and imaged using a LiCor Odyssey Fc. Membranes were washed with TBS-T and stained using Coomassie blue for 30 seconds, followed by destaining with water. The membranes were dried and imaged for total protein, imaged together to show total lanes.