Fig 2: Proposed model for the role of FES in neutrophil phagocytosis via SYK-HS1-PLCγ2 pathway activation.Representations are simplified for illustration purposes.

Fig 3: FESS700C can be visualized by fluorescent probe WEL033.a Design of fluorescent probe WEL033 (Cy5-conjugate). b Dose-dependent labeling of full-length FESS700C by WEL033 in HEK293T cell lysate. c Labeling by WEL033 is specific, exclusive for FESS700C and dependent on catalytic lysine 590. Lysates were preincubated with vehicle or 4 and labeled by WEL033 (250 nM). d, e Visualization of FESS700C target engagement by WEL028 and TAE684. Lysate was preincubated with WEL028 or TAE684 and labeled by WEL033 (250 nM). Band intensities were normalized to vehicle-treated control (n = 3). f, g WEL028 engages recombinantly expressed FESS700C in live cells. Transfected HEK293T cells were treated with WEL028 and labeled proteins were visualized using click chemistry. Band intensities were normalized to highest concentration (n = 3). h, i WEL028 blocks FESS700C but not FESWT autophosphorylation (visualized by immunoblot using anti-phospho-FES Y713). Band intensities were normalized to vehicle-treated control (n = 3). Data represent means ± SEM. Statistical analysis: two-tailed t-test: *** P < 0.001. Source data are provided as a Source Data file.

Fig 4: FESS700C is selectively and covalently inhibited by WEL028.a Proposed covalent binding mode of compound 4 to Cys700 in crystal structure of FES (PDB code: 4e93). b In vitro inhibition profile of FESWT and FESS700C by WEL028 (TR-FRET assay, n = 3). c Inhibition profile of WEL028 (100 nM) on FESWT and FESS700C on peptide substrates (PamChip® microarray, normalized to vehicle-treated control, n = 3). Scale shows residual activity from minimum (0%, red) to maximum (100%, gray). d MS/MS-based identification of WEL028 covalently bound to Cys700. Precursor ion (m/z [M + 2H]2+ = 689.2671) was fragmented and signature ions are shown. Precursor ion was not observed in vehicle-treated control. e In vitro selectivity profile of WEL028 (1 µM, 1 h preincubation) on 380 recombinant kinases, visualized as waterfall plot (each data point is an individual kinase; n = 2). Data represent means ± SEM. Source data are provided as a Source Data file.

Fig 5: Design and characterization of FES cysteine point mutants.a Location of mutated active-site residues (green) in FES crystal structure with bound reversible inhibitor TAE684 (PDB code: 4e93). b Activity of recombinantly expressed FES mutants compared to WT, determined as relative amount of phosphorylated peptide substrate after 60 min incubation using TR-FRET assay. c Reaction progress kinetics for FESWT and FESS700C. d Determination of ATP KM for FESWT and FESS700C. Enzyme reactions in TR-FRET assay were performed with ULight-TK peptide (50 nM) and ATP (b, c: 100 µM, d: variable) and quenched (b, d: after 60 min, c: variable). e Peptide phosphorylation substrate profile for FESWT and FESS700C as determined in PamChip® microarray. Peptides were filtered for those with ATP-dependent signal and log2 of signal intensity >3. The peptide substrates were identical for FESWT and FESS700C (Venn diagram, inset). f Preferred substrate consensus sequence based on FESWT substrate profile. Illustration was rendered using Enologos (http://www.benoslab.pitt.edu). g SH2 domain binding profile for FESWT and FESS700C as determined in PamChip® microarray. Peptides with non-specific antibody binding were excluded. The peptide SH2 binding partners were identical for FESWT and FESS700C (Venn diagram, inset). Data represent means ± SEM (n = 3). Statistical analysis: ANOVA with Holm–Sidak’s multiple comparisons correction: *** P < 0.001; NS if P > 0.05. Source data are provided as a Source Data file.