Fig. 5: SEE-CITE and functional studies confirm activity and labelling sites for asciminib binders.

a, General SEE-CITE-based AfBPP workflow. b, Scatter plots of peptides labelled by 4c (100 μM, 1 h) and 5c (100 μM, 1 h) in K562 cells. Data are average log₂(H/L) values (n = 2 biological replicates) including singletons imputed using H/L ratio of 20. Coloured dots indicate probe-modified peptides for selected high-value targets. c, Heatmap comparing average log₂(H/L) values for SEE-CITE-identified sites of labelling for selected targets in b and those in K562 cells labelled with 5c (20 μM, 1 h) or 3 (4 μM, 1 h). d,e, In-gel fluorescence analyses of HEK293T cells overexpressing wild-type (WT) RTN4, mutant C1101S-RTN4 labelled with 5c (100 μM, 1 h) (d) or in a competitive manner with asciminib (0.5 h) and 5b (1 μM, 0.5 h) (e) with GFP-HEK293T cell line as a control. f, A schematic representation of NOGO66–RTNR interaction, assayed with AP fusion proteins. g,h, Quantification of Nogo66–RTNR interaction via AP binding assays in HEK293T cells stably overexpressing RTNR incubated with AP fusion proteins pre-treated with asciminib (10 μM, 15 min) (g) or overexpressing GFP or RTNR incubated with AP, wild-type (WT) Nogo66 or C1101W-Nogo66 proteins (h). i, Docking of asciminib into the SEE-CITE COX5A labelling region (T79-Y82). j–l, In-gel fluorescence analyses of HEK293T cells transiently overexpressing FLAG-tagged GFP, wild-type COX5A or mutant COX5A in a competitive manner with asciminib and/or dasatinib (1 h) and 5b (j) or 5c (k) (10 μM, 1 h) or in a UV-dependent manner with 5c (10 μM, 1 h) (l). m, Immunoblot analysis of HEK293T cells transiently overexpressing FLAG-tagged GFP or COX5A (WT and mutants). n, FLAG immunoprecipitation (IP) with HEK293T transiently overexpressing FLAG-tagged GFP or COX5A (WT and mutants). o, State 3 respiration in plasma membrane-permeabilized HeLa cells stably overexpressing GFP–HA or COX5A–HA with or without asciminib treatment (10 μM, 4 h). Cells were offered pyruvate/malate as respiratory substrates. p, A schematic representation depicting effects of asciminib on COX5A and mitochondria functions. For g, h and o, data presented are mean ± s.d., with P values calculated using two-tailed unpaired and paired Student’s t-tests, respectively, using n = 3 biological replicates (g and h) and n = 6 biological replicates (o) (from left to right: P = 0.651571 and 0.000313 (g); P = 0.019546, 0.402216, 0.018749, 0.000398, 0.000959 and 0.000051 (h); P = 0.0461, 0.180008, 0.270314 and 0.007380 (o)) with *P < 0.05, **P < 0.01,***P < 0.001, and NS (not significant) P > 0.05. For d, e and j–m, data are presented for n = 3 independent experiments. NS, not significant; WT, wild type; OE, overexpression. All MS data can be found in Supplementary Table 9. Illustrations in a, f and p created in BioRender; Ngo, C. https://biorender.com/bxsja9h (2026).

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