Fig. 4: Detection of matriglycan on HAP1-DAG1- cells by flow cytometry. | Nature Communications

Fig. 4: Detection of matriglycan on HAP1-DAG1- cells by flow cytometry.

From: Cell surface glycan engineering reveals that matriglycan alone can recapitulate dystroglycan binding and function

Fig. 4

a CMP-Neu5Ac’s modified with defined matriglycan polymeric repeats (100 μM) are engineered on HAP1-DAG1- cells using ST6GAL1 in the presence of C. perfringens neuraminidase. b Detection of matriglycan with 1, 4, 5, and 6 disaccharide repeats on HAP1-DAG1- cells by flow cytometry. Cells were stained with avidin-AF488 and co-stained with PI to exclude non-viable cells. c Detection of matriglycan with 4 repeats at various concentrations of modified donor. d Binding of IIH6 to HAP1-WT and HAP1-DAG1- matriglycan modified cells. e Shotgun proteomics analysis of proteins immunoprecipitated from HAP1-DAG1- cells labeled with Biotin or Biotin+8 disaccharide repeats. Proteins present in the negative control experiment (unlabelled cells), had fewer than 10 spectral counts in the CMP-Neu5Ac-(Biotin) labeling experiment, or known to be localized in intracellular compartments as assessed by UNIPROT annotations, were excluded. Proteins shown are all annotated in UNIPROT to contain sites of N-glycosylation or were manually validated to contain at least one N-X-(S/T) N-glycosylation sequon in the primary sequence. One representative run is shown. Three independent experiments were performed with similar results each time. Source data are provided as a Source Data file.

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