Fig. 4

The abundances of Dpagt1 and the NgBR are influenced by O-GlcNAc (A) Western blot of O-GlcNAc modified proteins show treatment with 16 ?M OSMI-1 reduces global O-GlcNAc levels in 6-dpf pmm2m/m mutant larvae. See Figure S4 for Ponceau gel. ROI1?3 represent three ROIs quantitated. (B) Graphs show quantitation of O-GlcNAc levels in three ROIs comparing DMSO or OSMI-treated mutant larvae. n = 5 biological replicates per genotype per condition with 10 embryos per sample. (C) Western blot comparing Dpagt1 and NgBR levels in WT and mutant larvae treated with OSMI-1 shows globally reducing protein O-GlcNAc levels corresponding to a reduction in their protein abundance. (D) Graphs comparing protein abundance of either Dpagt1 or the NgBR in OSMI-1-treated and untreated mutant larvae. See Figure S4 for graphs of WT quantitation and relative values normalized to Ponceau staining. n = 4?5 biological replicates per genotype per condition, each with 10 embryos per sample. (E) qRT-PCR analyses of transcript abundance of show no reduction after OSMI-1 treatment in any gene assayed 6 or 7 dpf. n = 3 biological replicates per genotype per condition with 10 embryos per sample. (F) Western blot of protein O-GlcNAc levels in Thiamet G-treated WT larvae. (G) Graph showing Thiamet G increases O-GlcNAc levels nearly 2-fold. n = 4 biological replicates per condition with 10 embryos per sample. (H) Western blot of DMSO or Thiamet G-treated WT shows increased O-GlcNAc levels correspond to an increase in the protein abundance of both Dpagt1 and the NgBR, with changes in Gfpt and Alg9 more variable. (I) Graphs show Dpagt1, NgBR, Gfpt, and Alg9 protein abundance in DMSO- or Thiamet G-treated animals. n = 3 biological replicates per condition. (J) PLAs performed on sections of WT and pmm2m/m mutant larvae 8 dpf show reactivity27 indicating both proteins may be O-GlcNAcylated. Reactivity is increased in mutants. Alg9 shows no reactivity. Scale bar, 10 ?m. (K) Graph shows the number of cells per embryo per genotype that exhibit reactivity indicating O-GlcNAcylation of either Dpagt1 or the NgBR. n = 7 embryos per genotype. (L) Graph shows the signal intensity (normalized to EGFP) of individual cells that exhibits PLA positive reactivity. n = 6 embryos per genotype. (M and N) Western analyses of immunoprecipitated fractions using either anti-Dpagt1 (M) or anti-NgBR (N) antibodies. Blots were probed for Dpagt1, the NgBR, or for O-GlcNAc (as indicated on the figure). Arrows highlight expected protein forms (red and green arrows) and non-specific bands (blue). (O) Mechanistic model showing a mechanism whereby increased UDP-GlcNAc levels drive O-GlcNAcylation of Dpagt1 and the NgBR in mutant larvae. For all experiments, error = SEM, Student?s t test, ?p < 0.05, ??p < 0.01, ????p < 0.0001. Where a red line and red star are shown, the Welch?s test was used due to sample variance. ?p < 0.05. See also Figure S4.