Split-NanoLuc cellular target engagement assay N-terminal HiBiT-SOCS2 fusion was generated by encoding SOCS2 (32-198) in pBiT3.1-N expression vector (Promega). The transfection and target engagement assay were performed by modifying a NanoLuc target engagement protocol (Promega) with some small modi8ications 68 . Brie8ly, HiBiT-SOCS2 fusion construct was transfected into HeLa cells using FuGENE HD (Promega) according to the manufacturer's protocol. HiBiT-SOCS2 fusion construct was diluted into Transfection Carrier DNA (Promega) at a mass ratio of 1:100 or 1:10 for permeabilised and live cell CETSA respectively. FuGENE HD was added at a ratio of 1:3 (μg DNA: μL FuGENE HD). 1 part (vol) of FuGENE HD complexes thus formed were combined with 20 parts (vol) of HeLa cells suspended at a density of 2 x 10 5 cells/mL, followed by incubation in a humidi8ied, 37 °C + 5% CO2 incubator. After 20 h of incubation HeLa cells were detached and resuspended at a density of 2.5 x 10 5 cells/mL in Opti-MEM media without serum or phenol red but supplemented with protease inhibitor cocktail (Roche). Compounds stocks were added to cell suspension to maintain a 8inal DMSO concentration of 1%. For the permeabilised assay format, 96-well PCR plates (Thermo) were pre-treated with digitonin to have a 8inal well concentration of 50 μg/ml. 50 μl Opti-MEM cell suspension was then aliquoted to each well of the 96-well plate. The plates are then covered with microporous tape sheet (Qiagen) prior to incubation in a humidi8ied, 37 °C + 5% CO2 incubator. Postincubation we heat the PCR plates in 16-point temperature (split across two runs) curve spanning degrees 40-72 °C temperature span (CFX96 Touch Real-Time PCR Detection System, Bio-Rad). The thermal cycler block was allowed to rise to the designated temperatures prior to placement of the PCR plates into the heating block and heat treatment for 3 minutes. After incubating the plates at room temperature for over 3 minutes, 50ul of the Nano-Glo HiBiT Lytic mix (Promega) was added to each well. Samples were mixed by pipetting and placing the plate on an orbital shaker (600rpm) for 5 minutes. 20 μl of the samples (in triplicates) were transferred to a AlphaPlate-384 plate (Perkin Elmer). Luminescence was recorded with GloMax Discover Microplate Reader (Promega). To generate apparent Tagg curves, the data is 8irst converted to percent stabilized by relating the observed luminescence to the luminescence of the lowest temperature (40 °C) for that given sample. Data are then 8itted to obtain apparent Tagg values using the Boltzmann Sigmoid equation using Graphpad Prism software.