VER2 and O-GlcNAc Signaling Figure 8. Immunogold labeling of VER2-GFP and colocalization of VER2-GFP with VSRAt in Arabidopsis leaves. (A)–(D) Immunogold labeling of VER2-GFP. Sections were incubated with anti-GFP antibody (A to C) or preimmune serum (D) followed by gold-conjugated secondary antibody. Gold particles accumulate on ER (A) Golgi apparatus (B) and PVC (C). No specific labeling was found in the control image (D). ER is indicated with arrows. Bars, 0.2 mm. (E) VER2-GFP fusion signals with ring structures. (F)–(H) Colocalization of VER2-GFP with VSRAt. The anti-VSR-marked prevacuolar organelles are shown in red channel. Endogenous VSR was detected by indirect immunofluorescence labeling. Fixed leaves were stained with anti-VSR antibody followed by a TRITC-labeled anti-rabbit antibody (F). GFP signals indicated punctate distribution of VER2-GFP in green channel observed from the fixed leaves (G). Merged image of (F) and (G) is shown as (H). g, Golgi; cw, cell wall. Bars, 20 mm. doi:10.1371/journal.pone.0004854.g008 fructose, maltose, and albumin egg, was serially diluted with a starting concentration of 200 mM and tested for carbohydrate- binding specificity. Solutions containing 10 ml of purified VER2 and 10 ml sugars or glycoproteins were preincubated for 1 h at room temperature, then 20 ml of a 2% rabbit erythrocyte suspension was added, and the agglutination was evaluated after 1 h. Production and purification of peptide-specific antibody A peptide of 12 amino acids (KRRTTDSRGGGN, amino acids 205 to 214) predicted to be highly specific to VER2 was synthesized [32]. The peptide was linked to BSA via its N- terminal lysine, and the conjugate was used to immunize rabbits. To remove non-specific antibodies that might react with the BSA Figure 9. Location patterns of VER2-S33G and VER2-T209A mutations fused to GFP. (A) VER2-S33G mutation. (B) VER2-T209A mutation. Bars, 20 mm. doi:10.1371/journal.pone.0004854.g009 PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4854