Extended Data Fig. 3 |. Confirming direct engagement of NONO_C145 by active compounds using alkyne probe 14.

a, Structure of alkyne probe 14. b, Western blots showing effects of alkyne probe 14 on AR-FL and AR-V7 protein content of 22Rv1 cells. Active compounds 5 and (R)-SKBG-1 and inactive control compounds 8 and (S)-SKBG-1 are shown for comparison. Cells were treated with 20 μM of each compound for 24 h. Also shown are western blotting signals for PSPC1. Data are from a single experiment representative of two independent experiments. c, Waterfall plot showing global protein enrichment profiles from MS-ABPP experiments performed with alkyne 14-treated sgNONO 22Rv1 cells expressing FLAG-WT- or C145S-NONO. Cells were treated with 20 μM of 14 for 1 h prior to lysis and enrichment and quantification of 14-labeled proteins (see Methods section for more details). Data are presented as the ratio of enrichment of proteins from WT-NONO/C145S-NONO cells. Data are average values for all quantified peptides for each protein from two independent replicates. d, Gel-based ABPP experiment showing that alkyne 14 labels WT-NONO, but not C145S-NONO expressed as FLAG epitope-tagged proteins in sgNONO 22Rv1 cells. sgNONO cells (with or without exogenous expression of WT- or C145S-NONO) were pretreated with DMSO or (R)-SKBG-1 (20 μM, 2 h) followed by DMSO or alkyne 14 (20 μM, 1 h), lysed, conjugated to a rhodamine-azide (Rh-N₃) by copper-catalyzed azide-alkyne cycloaddition chemistry, and analyzed by SDS-PAGE and in-gel fluorescence. e, Concentration-dependent effects of (R)-SKBG-1 and (S)-SKBG-1 on alkyne 14 labeling of FLAG-tagged WT-NONO expressed in HEK cells. Cells were treated with (R)-SKBG-1 or (S)-SKBG-1 for 2 h, followed by 14 (20 μM, 1 h), and then processed as described in d. Mock (empty vector-transfected) cells and cells expressing FLAG-tagged C145S-NONO are shown as controls. For d and e, red asterisk marks band corresponding to the MW of FLAG-NONO. IB, immunoblot. f, Quantification of blockade of 14 labeling of recombinant FLAG-WT-NONO by (R)-SKBG-1. Data are mean values ± s.e.m. for four independent experiments. 95% confidence interval or calculated IC₅₀ value is shown in brackets.