Fig. 6. The X409 domain mediates the mucin binding of StcE.

a Graphic depiction of the design of enzyme activity and binding assays performed. b Schematic representation of expression construct designs for full coding StcE^WT, StcE^E447D, X409 domain truncated StcE (StcE^ΔX409), and the isolated X409 domains ± GFP (left). Flow cytometry analysis of cleavage (dose titration) of membrane-bound MUC2#1 expressed in HEK293^WT cells by StcE^WT, StcE^E447D, StcE^ΔX409, and the X409 domain detected by anti-FLAG staining and flow cytometry (right). Representative data of three independent experiments with similar results are shown. c Immunofluorescense staining of representative sections of normal colon stained with StcE, StcE^E447D, StcE^ΔX409, X409, and anti-MUC2 mAb (PMH1). Counterstained with DAPI (blue). Scale bars = 100 μm. d Flow cytometry analysis of X409-GFP binding (0–10 μg/mL dose titration) to HEK293^WT cells stably expressing mucin TR reporters tagged with CFP (instead of GFP). Average MFI ± SEM values from two independent experiments are shown. e Flow cytometry analysis of X409-GFP binding (1 µg/mL) to MUC2#1, MUC5AC, MUC7, and MUC1 reporters expressed on glycoengineered HEK293 cells (core2, dST, mSTa, T, Tn, core3, and STn). Data are presented as average MFI ± SEM of two (MUC1) or four (other MUCs) independent experiments. Source data are provided as a Source Data file.