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. 2021 Apr 15;22(6):e51649. doi: 10.15252/embr.202051649

Figure 4. Identification of O‐GlcNAcylation sites of TDP‐43.

Figure 4

  1. TDP‐43 interacts with OGT examined by immunoblotting.
  2. In vitro glycosylation assay was performed using immunoprecipitated OGT incubated with recombinant His‐tagged TDP‐43 in the presence of UDP‐GlcNAc. The O‐GlcNAcylated TDP‐43 protein was detected by immunoblotting.
  3. Sequence alignment of TDP‐43 segments containing the identified O‐GlcNAcylation sites in different species. The amino acids with red color display the conserved O‐GlcNAcylated sites.
  4. Structural modeling of the RRM2 domain bound to a single strand DNA illustrates the O‐GlcNAcylation sites T199 (pink) and T233 (red) of TDP‐43 with surrounded DNA (light orange).
  5. In vitro glycosylation assays were performed using immunoprecipitated Flag‐OGT incubated with recombinant GST‐tagged TDP‐43 fragments (a.a. 102‐269) in the presence of UDP‐GlcNAc. The O‐GlcNAcylated TDP‐43 proteins were detected by immunoblotting.
  6. Quantification of (E). Mean ± SD, unpaired two‐tailed t‐test, n = 4 biological replicates. ****P < 10−4.
  7. Cells expressing the indicated forms of GFP‐TDP‐43 were subjected to immunoprecipitation, and the O‐GlcNAcylated levels of TDP‐43 were examined.
  8. Quantification of (G). Mean ± SD, unpaired two‐tailed t‐test, n = 3 biological replicates. *P < 0.05, ****P < 10−4, n.s. not significant.

Source data are available online for this figure.