Figure 2. TAB3 is O-GlcNAcylated on Ser408 in TNBC cells.

(A) Prokaryotic expressed TAB3 was incubated with recombinant hOGT. The samples were subjected to SDS-PAGE and immunoblotted with a generic O-GlcNAc antibody CTD110.6. Total protein was detected with the Flag antibody as a loading control. (B) Chemoenzymatic labeling of the O-GlcNAc residue In vitro O-GlcNAcylated TAB1 was subjected to enzymatic labeling using galactosyltransferase (mGalT1) labeling and UDP-GalNAz, before reacting with biotin alkyne for detection with HRP. The samples were subjected to SDS-PAGE and probed with horseradish peroxidase conjugated streptavidin (Extravidin-HRP). (C) Lysates from HEK293 cells transfected with cmv-Flag or Flag-TAB3 were immunoprecipitated for Flag antibody and treated with β-elimination. The samples were subjected to SDS-PAGE and probed with CTD110.6 antibody. (D) In vivo O-GlcNAcylation of TAB3 was detected by immunoprecipitating the endogenous TAB3 from MDA-MB-231 cells (treated with or without 1 mM GlcNAcstatin) using an antibody directed against TAB3. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted with CTD110.6 and then with the TAB3 antibody for loading controls. (E) An ETD MS/MS spectrum recorded on [M + 4H]⁺⁴ ions of GlcNAcylated peptide QHSLYTATTPPSSSPSR. An ETD-enabled LTQ mass spectrometer was operated followed by four data-dependent scans after every MS1 scan to generate the ETD spectrum, which is presented as a subtracted spectrum. Predicted product c′- and z′- ions are listed above and below the peptide sequence, respectively. Single- and double-charged ions are listed as monoisotopic and average masses, respectively. Observed product ions are underlined and are sufficient to define the O-GlcNAc residue at Ser-408. (F) MDA-MB-231 cells were cotransfected with FLAG-tagged wild-type or S408A mutant TAB3 along with OGT siRNA vector for 48 h. TAB3 was enriched and the O-GlcNAcylation of TAB3 was detected using antibody CTD110.6.