Fig. 2: SICFA allows determination of drug induced stability shifts in living cells.
a Schematic overview of the experimental workflow. Cells are treated with either vehicle (control) or ligand (drug) and divided into aliquots. Each aliquot is subjected to solvent-induced fixation at low concentrations of a fixative solution. After partial fixation, cells were lysed, and the lysate was centrifuged to separate the soluble fractions from the precipitate. The supernatant containing soluble proteins was collected for downstream analysis. Protein abundances were measured via immunoblotting for target verification or LC-MS/MS-based quantitative proteomics for target identification. This approach evaluates stability shifts of drug targets and downstream effector proteins within cells following drug treatment. b Western blot analysis validating the stabilization of TYMS by Raltitrexed in Jurkat cells at various fixative concentrations, with Tubulin as a loading control (n = 2 biological replicates). c Volcano plot illustrating the identification of raltitrexed targets using SICFA. The dashed horizontal line represents a −log10(p value) of 4.0. Known targets are highlighted in red. Source data are provided as a Source Data file. d Volcano plot showing target recognition by SICFA for dasatinib. The dashed horizontal line represents a −log10(p value) of 3.5. Known targets (red) and potential targets (green) are highlighted. Source data are provided as a Source Data file. e Volcano plot showing the targets of MTX (20 μM, 30 min incubation in cell lysate) in the SIP experiment. Known target is highlighted. Source data are provided as a Source Data file. f Volcano plot showing the targets of MTX (1 μM, 30 min incubation in live cells) in the SICFA experiment. Known target (red) and potential targets (green) are highlighted. c–f p values from two-sided empirical Bayes t tests without adjustment, n = 5 cell replicates. Source data are provided as a Source Data file. g Schematic representation of metabolic pathways influenced by MTX, highlighting interactions with target proteins TYMS and DHFR.
