Fig. 4: Characterisation of cellular GSK3 degrader probes.

a Endogenous GSK3 paralog degradation with compounds 24 and 26 and negative controls 27 and 28 in HEK293 cells (representative blots of n = 3 biological replicates, 2–4 h). The samples derive from the same experiment, gels and blots were processed in parallel. b, c Immunoblots shown in panel A were quantified, normalised to relevant internal controls and plotted for changes in GSK3β and GSK3α levels. Compound 27 in light grey, 24 in green, 28 in dark grey, and 26 in blue, with shading intensity corresponding to concentration (lightest = 10 nM, darkest = 30 nM) (n = 3 biological replicates with error bars representing mean and ± SD, p values are calculated as <0.0001, two-tailed unpaired t-test (GraphPad)). d Mechanism of action studies in HEK293 cells for compounds 24 and 26 showing rescue with 2 h pre-treatment of proteasome inhibitor (1 µM, MG132), neddylation inhibitor (1 µM, MLN4924) and GSK3 inhibitor (1 µM, 29). Sample processing controls run on different gels in parallel (Representative blot of n = 3 biological replicates). e, f Immunoblots shown in (d) were quantified, normalised to relevant internal controls and plotted for changes in GSK3β and GSK3α levels, 24 in green and 26 in blue (n = 3 biological replicates with error bars representing mean and ± SD). g Evaluation of changes in phosphorylation levels of GSK3β following compound 24, 27 or GSK3 inhibitor (29) treatments for 30 and 120 min in HEK293 cells (n = 3 biological replicates). The samples derive from the same experiment, gels and blots were processed in parallel. h Dose dependent effect of compound 24 on GSK3 substrates in differentiated SH-SY5Y cells (representative of n = 3 biological replicates, at indicated concentrations, 24 h). The samples derive from the same experiment, gels and blots were processed in parallel.