Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins

doi: 10.1073/pnas.1813020116

Figure Lengend Snippet: StcE increased the number of assigned glycosites, number of localized glycans, and sequence coverage of every protein studied. (A) Recombinant substrates were digested with StcE, de–N-glycosylated with PNGaseF, trypsinized, and then subjected to MS using an HCD-triggered ETD instrument method. ETD spectra were used to assign glycosites. (B, Top) ETD spectra of N- and C-terminal StcE-cleaved peptides LSTMMSPTT and STNASTVPFR from CD43 from the experiment described in A. (B, Bottom) ETD spectrum from the control sample (PNGaseF and trypsin only) is shown. The lowercase “n” denotes deamidation. Parentheses indicate that the sites modified with GalNAc residues could not be assigned. Rel., relative.

Article Snippet: Recombinantly expressed CD43 was purchased from Origene (TP304195).

Techniques: Sequencing, Recombinant, Control, Modification

Journal: The Journal of Cell Biology

Article Title: Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

doi: 10.1083/jcb.200707199

Figure Lengend Snippet: CD43 specifically coimmunoprecipitates with moesin. (A and B) Moesin (A) and ezrin (B) immunoprecipitates from resting and activated Jurkat T cells immunoblotted with CD43 and moesin or ezrin antibodies. (C) Resting and 1-min–activated Jurkat T cells were fixed and stained for moesin (green) and CD43 (red).

Article Snippet: R-phycoerythrin–labeled mouse anti–human CD3 and mouse anti–human CD43 were obtained from BD Biosciences.

Techniques: Staining

Journal: Cellular and Molecular Life Sciences

Article Title: Tumor cell-intrinsic and tumor microenvironmental conditions co-determine signaling by the glycoimmune checkpoint receptor Siglec-7

doi: 10.1007/s00018-023-04816-6

Figure Lengend Snippet: CD43-expressing cells can induce chSiglec-7 signaling under the condition of sufficient sialic acid availability. a HEK293-6E cells were transfected to express CD43 or were mock-transfected and CD43 expression was determined by flow cytometry and compared to the respective isotype control. b Siglec-7 ligand expression was determined by a recombinant human Siglec-7 Fc staining, measured by flow cytometry. c chSiglec-7 or d chSiglec-7R124A expressing Jurkat/MA cells were co-cultured (1:1) with the CD43 or mock transfected HEK293-6E cells and luminescence was assessed using a luciferase assay (n = 5). c , d Nested data are presented of 4 experiments, each performed in duplicate. e Siglec-7 Fc binding to parental IMR-32 cells or CD43 + IMR-32 cells was assessed by flow cytometry and the MFI was quantified. f chSiglec-7 or g chSiglec-7R124A expressing Jurkat/MA cells were co-cultured (1:1) with parental IMR-32 cells or CD43 + IMR-32 cells and luminescence as assessed using a luciferase assay (n = 2). IMR-32 cells were pretreated with Ac 5 Neu5Ac or DMSO as vehicle control for 3 days, which was refreshed on day 2. Representative data are shown of two independent experiments and data present mean ± SD

Article Snippet: The open reading frame of human CD43 was cloned from a pGEM-T vector with the human CD43 open reading frame (SinoBiological, HG13108-G) into the pEGFP-N3 vector, replacing EGFP.

Techniques: Expressing, Transfection, Flow Cytometry, Recombinant, Staining, Cell Culture, Luciferase, Binding Assay