Fig. 1: JQ1–protein interactions studied by Drug-ID.

a, General scheme. After induced expression of a biotin ligase–Halo fusion, the Halo-tag covalently binds to a CA-modified probe. Addition of biotin will lead to the formation of biotin–AMP, which reacts with free lysine residues in close proximity to the probe. b, Expression and activity of the biotin ligase NLS-Halo-eGFP-BASU integrated into Flp-In T-REx 293 cells. Transgene expression was induced with doxycycline and followed by eGFP fluorescence (green channel). Cells were incubated with biotin (50 µM) for 0–24 h, and biotin deposition was stained with Atto594–streptavidin (red channel). Cell nuclei were stained with DAPI (blue channel) (n = 1). c, Structure of the JQ1-CA probe. d, Scheme of the passive uptake and conjugation assay of the JQ1-CA probe. e, PAGE analysis of JQ1-CA uptake into transgenic Flp-In T-REx 293 cells. Concentration-dependent conjugation of the JQ1-CA probe with the biotin ligase was visualized by conjugation of a competing fluorescent TMR-CA probe (red channel). Western blot against ACTB and Halo-tag served as loading controls (n = 1). f, Schematic overview of the SILAC–MS/MS experiment. g, SILAC–MS/MS for 500 nM JQ1-CA against DMSO. Plotted was the enrichment (log₂) of replicate 1 against replicate 2 with swapped SILAC labeling. h, SILAC–MS/MS for the competition experiment 500 nM JQ1-CA against 500 nM JQ1-CA + 5 µM JQ1. The enrichment with JQ1-CA over DMSO is displayed on the x axis, and the depletion upon addition of JQ1 in 10-fold excess is displayed on the y axis (JQ1-CA + JQ1/JQ1-CA). Plotted was the enrichment of replicate 1 against replicate 2 with swapped SILAC labels. Statistical significance of hits in g and h was calculated with respect to the distance of the median of the distribution of all protein ratios as well as protein intensities using Perseus. Threshold for significance was set to P < 0.01 for all MS/MS experiments. Dox, doxycycline; rep, replicate.

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