Journal: The Journal of Biological Chemistry

Article Title: N -Glycans on EGF domain-specific O -GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization

doi: 10.1074/jbc.RA119.012280

Figure Lengend Snippet: EOGT is modified with oligomannose N-glycans. A, lectin blot analysis of FLAG-EOGT isoforms. HEK293T cells were transfected to express each FLAG-EOGT isoform. The cell lysates were subjected to immunoprecipitation with FLAG-antibody followed by detection by biotinylated ConA lectin (ConA-biotin) or EOGT-CT antibody. An asterisk indicates nonspecific bands. B, LC–MS/MS spectra of glycopeptides modified with HexNAc2Hex9 N-glycan at N263 (top) and HexNAc2Hex7 N-glycan at Asn-354 (bottom) of FLAG-EOGT. Chymotryptic or tryptic glycopeptides prepared from recombinant FLAG-EOGT were analyzed by LC–MS/MS. Fragments ions corresponding to b and y ions, peptides with truncated glycans, and glycans are shown by arrows. Blue square, HexNAc (presumably GlcNAc); green circle, hexose (presumably mannose). C, bar graphs showing relative abundance of different N-glycan glycoforms at EOGT Asn-263 and Asn-354. D, endogenous EOGT sensitivity to Endo H digestion. HEK293T cell lysates were incubated in the absence or presence of Endo H and analyzed by immunoblotting with EOGT-specific AER61 antibody. Recombinant FLAG-EOGT and FLAG-EOGTN263Q/N354Q were analyzed in parallel as controls.

Article Snippet: Next, the beads were washed extensively with the lysis buffer and eluted with 50 µl of 3× FLAG peptides (1 µg/µl, MBL) in 10 m m HEPES, pH 7.0.

Techniques: Modification, Transfection, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Recombinant, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: N -Glycans on EGF domain-specific O -GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization

doi: 10.1074/jbc.RA119.012280

Figure Lengend Snippet: Reduced O-GlcNAc stoichiometry on Notch1 in HEK293T cells expressing N-glycan-deficient EOGT. A, MS analysis of tryptic glycopeptides prepared from FLAG-Notch1-TM harboring O-GlcNAcylation sites. FLAG-Notch1-TM was expressed in EOGT-deficient HEK293T cells exogenously expressing WT or the N263Q/N354Q EOGT. EICs show the relative signal intensity corresponding to the peptides without modification (orange) or with O-GlcNAc (blue) on EGF2, EGF10, EGF11, EGF21, and EGF23 of Notch1. Note that no elongated O-GlcNAc glycoforms were detected. Data are presented as mean ± S.D. (n = 2). LC–MS/MS spectra of tryptic glycopeptides are shown in Fig. S3. B, quantification of O-GlcNAc glycans on representative EGF domains. EIC peak heights were measured and expressed as percent area. The color code is same as described in A.

Article Snippet: Next, the beads were washed extensively with the lysis buffer and eluted with 50 µl of 3× FLAG peptides (1 µg/µl, MBL) in 10 m m HEPES, pH 7.0.

Techniques: Expressing, Modification, Liquid Chromatography with Mass Spectroscopy