Figure 4.

NSL3 can be O-GlcNAcylated by OGT1 in vitro and in vivo. A, O-GlcNAcylation of stably expressed FLAG-NSL3 and its associated proteins. Anti-FLAG-agarose eluates from FLAG-NSL3-expressing HEK293FRT cells and parental 293FRT cells (as a negative control) were subjected to SDS-PAGE in a 4–20% gradient gel, and O-GlcNAc-modified proteins were visualized by Western blotting with anti-GlcNAc antibody. B, confirmation of O-GlcNAc transferase activity of the recombinant OGT1. Insect cell-expressed/purified full-length OGT1 was visualized by Coomassie Brilliant Blue staining (upper). An in vitro O-GlcNAc transferase assay was performed by mixing recombinant OGT1, UDP-[³H]GlcNAc, and Nup62. O-GlcNAc transferase activity was measured by autoradiography (lower). C, O-GlcNAcylation of recombinant NSL3 by OGT1. Insect cell-expressed/purified full-length NSL3 (FL-NSL3) (upper) was used in an O-GlcNAc transferase assay. Modified O-GlcNAcylation was measured by anti-GlcNAc antibody (lower). D, OGA antagonistic effect on OGT1-produced O-GlcNAcylation of NSL3. O-GlcNAc transferase assays were performed as indicated in the presence or absence of OGA. Modified O-GlcNAcylation was visualized by autoradiography (upper panel) or by Western blotting with anti-GlcNAc antibody (lower panel). E, WGA lectin affinity purification. Whole-cell lysates (WCL) from 293T cells (upper panel) were subjected to WGA affinity purification. Binding reactions were as indicated. The precipitates were then analyzed by Western blotting for endogenous NSL3. Free GlcNAc (20 mm) was added for control of specificity (lower panel). Lanes 3 and 4 and lanes 5 and 6 represent two biological experimental repeats. Input 1 and Input 2 indicate whole-cell lysates harvested at different times. F, WGA lectin affinity purification in FLAG-OGA transfected cells. Prepared whole-cell lysates of FLAG-OGA (5 or 10 μg/10-cm dish) transiently transfected (48 h) 293T cells were subjected to WGA affinity purification. Endogenous O-GlcNAcylated NSL3 was detected by Western blotting with NSL3 antibody. IB, immunoblot; Fl, FLAG.