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Fig. 6 NgBR abundance and O-GlcNAcylation are increased in the brains of pmm2m/m mutants (A) Western blot analyses of isolated brains show only abundance of the NgBR, but not Dpagt1 or Alg9, is increased in the mutant brain. (B) Graphs showing quantitation of protein abundances reveal 4- to 5-fold increases in the NgBR are matched in the mutant brain by variable Dpagt1 levels and a reduction in Alg9. n = 3 biological replicates per genotype per sample. (C) Western analyses of protein O-GlcNAcylation show no significant global changes in overall modification of brains proteins. (D) Graph quantitating O-GlcNAc shows little difference between WT and mutant brains. Abundance is normalized to Ponceau stains. n = 3 biological replicates per genotype per time point. (E and F) PLAs of NgBR and O-GlcNAc (E) or Dpagt1 and O-GlcNAc (F) interactions performed on sections of fli1a:EGFP-positive brains show O-GlcNAcylation of each protein is increased in the brains of pmm2m/m mutants. Boxed areas shown in lower panels highlight increased PLA reactivity.27 Blue, DAPI-stained nuclei. (G) Graphs show PLA signal intensity, scored as total pixel intensity across entire brain. n = 3?4 brains per genotype per condition. All scale bars, 20 ?m. (H) Western blot of O-GlcNAc (relative to Ponceau stain) in brains of DMSO- or Thiamet G-treated larvae. (I) Graph of quantitation of total O-GlcNAc signal normalized to Ponceau. n = 3 biological replicates per condition. (J) Western blot of NgBR, Dpagt1, and Alg9 abundance in brains of Thiamet G-treated larvae. (K) Graph showing quantitation of total NgBR, Dpagt1, or Alg9 abundance (normalized to levels in untreated). n = 3 biological replicates per condition. For all experiments, error = SEM, Student?s t test,?p < 0.05, ??p < 0.01, ???p < 0.001. See also Figure S6.