domain of the conjugate, the crude serum was first absorbed with CNBr-activated sepharose 4B resin (Amersham) to which BSA had covalently attached. The peptide-specific antibody was then purified with the resin and used for immunocytochemical localization of VER2 in wheat. Immunocytochemistry Plumules were fixed in 4% formaldehyde and 1% glutaralde- hyde in phosphate buffered saline (PBS, 0.1 mol/L Na2HPO4, NaH2PO4, pH 7.4) at 4uC for 4 h. Materials were then washed in PBS and dehydrated in ethanol, then 10-mm transverse sections of paraplast (Sigma) were prepared. Prior to incubation with the primary antibody, the sections were blocked with 1% (w/v) BSA in PBS at 37uC for 30 min. Purified peptide antibody was diluted 1:50 in PBS supplemented with BSA and applied to sections at 37uC for 1 h. The sections were washed 3 times for 10 min each with 0.05% (v/v) Tween 20 in PBS (PBST, pH 7.4) and incubated with the secondary antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG (diluted 1:500 in PBST), at 37uC for 1 h, then washed in PBST and distilled water. The alkaline phosphatase activity was detected by use of 4-nitro blue tetrazoliu chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), accord- ing to the manufacturer’s instructions (SABC). In control experiments, the primary antibody was replaced with preimmune rabbit serum. Sections were photographed before or after being mounted with 50% glycerol, under bright-field microscopy. The nuclei were indicated by hematoxylin staining. SDS-PAGE and western blot analysis Proteins were separated on 12% SDS-PAGE and electroblotted onto nitrocellulose sheets. Membranes were blocked for 1 h with 5% BSA in TBS-Tween buffer (Tris-HCL 20 mM, NaCL 150 mM, and Tween 0.05%, pH 8.0). Immunoprobing of VER2 was conducted with the anti-VER2 polyclonal antibody for 2 h at 4uC at a dilution of 1:500 in TBS. Three washes of 5 min each were performed with TBS-Tween. An anti-rabbit IgG conjugated with alkaline phosphatase was used as the secondary antibody at 1:1000 dilution for 1 h at room temperature. Three washes of 5 min each were performed with TBS-Tween, and target proteins were visualized by incubating the membranes with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). For ECL analysis, the secondary antibody was conjugated with horseradish peroxidase (HRP), and the mem- brane was exposed to film. Transgenic Arabidopsis All Arabidopsis plants used were ecotype C24. Seedlings were grown in a greenhouse under 16 h light/8 h dark. The VER2 ORF from a full-length VER2 cDNA was amplified by PCR with the following primers: 59-CCGTCTAGAATGGCCAAATTC- CAGATTAC-39 (with an XbaI site) and 59-CCAGGTACC- GACCGTGTAAACACCAAATG-39 (with a KpnI site). The amplified fragment was digested with XbaI and KpnI and cloned into available sites of pBI121-GFP, which contains the GFP driven by a cauliflower mosaic virus (CaMV) 35S promoter. VER2 was located between 35S and GFP. The construct was verified by DNA sequencing. The pBI121 plasmids carrying 35S::VER2-GFP and 35S::GFP were transformed into Agrobacterium tumefaciens strain GV3101 separately, and the resulting bacteria were used to transform Arabidopsis thaliana ecotype C24. The transformants were selected on plates of Murashige and Skoog (MS) medium containing 50 mg/ml kanamycin. Before being transferred to the greenhouse, transformants were vernalized at 4uC for 3 days. The leaves of VER2 and O-GlcNAc Signaling transgenic plants were used to observe GFP fluorescence. For some confocal microscopy observations, leaves were counter- stained with propidium iodide (PI) (Sigma-Aldrich) to show nuclei. Isoelectric focusing and SDS-PAGE For 2-D electrophoresis, samples and immobiline drystrip gels (7 cm, pH 3–10, Amersham) were rehydrated in a solution containing 9 M urea, 0.5% CHAPS, 0.3% DTT, 0.5% pharmalyte and 0.002% bromophenol blue at 20uC for 12 h. The samples were then run on immobiline gels at 500 V for 0.5 h, then 1000 V for 0.5 h and, finally, 5000 V for 1.5 h. After isoelectrofocusing, gels were denatured first in a balance buffer (50 mM Tris-CL, pH 8.8, 6 M urea, 30% sucrose, 2% SDS, and 0.002% bromophenol blue) containing 1% DTT for 15 min, then transferred to a balance buffer containing 2.5% iodoacetamide for another 15 min. Finally, proteins were resolved by 12% SDS-PAGE. Immunoprecipitation For phosphatase treatment of VER2 protein, extracts of vernalization- and devernalization-treated wheat seedlings were immunoprecipitated by use of anti-VER2 antiserum. Phosphatase treatment followed the manufacturer’s instructions (Biolab). The treated proteins underwent western blot analysis to detect the VER2 protein. For analysis of O-GlcNAc modification of VER2 in vernaliza- tion- and devernalization-treated wheat seedlings, the monoclonal antibody against O-GlcNAc was used to immunoprecipitate O- GlcNAc-modified proteins. VER2 in immunoprecipitated proteins was detected by immnoblotting with the polyclonal anti-VER2 antibody. Confocal microscopy and propyzamid treatment of Arabidopsis leaves Confocal images were taken under a LSM 510 META confocal laser scanning microscope (ZEISS). Three-week-old Arabidopsis cotyledons were used to examine the effect of propyzamid on the distribution of VER2-GFP. The final concentration was 5 mM for propyzamid dissolved in DMSO. Mutagenesis The VER2-S33G and VER2-T209A mutants were generated using PCR method. The two mutant expression constructs of pBI121-VER2-S33G-GFP and pBI121-VER2-T209A-GFP were sequenced to ensure generating the desired mutation. Electron microscopy Leaf fragments (1 to 2 mm) of Arabidopsis plants transformed with 35S::VER2-GFP were fixed in a buffer containing 2% formaldehyde (in 0.1 M PBS, pH 7.4) and 0.5% glutaraldehyde at 4uC for 2 h. They were then rinsed and dehydrated in an ethanol series. Dehydrated materials were embedded in London Resin White (Sigma). Ultrathin sections were prepared and collected on nickel grids precoated with 0.25% formvar. For immunogold labeling, grids were incubated with GFP antibody (diluted 1:400), then gold-coupled goat anti-rabbit antibody (1:30). Control sections were treated with preimmune serum. Sections were stained with 2% uranyl and acetate and examined by use of a JEM-1230 transmission microscope (Japan) operating at 80 kV. Supporting Information Figure S1 Lane1, molecular weight markers; The protein markers transferred to nitrocellulose sheet were indicated by PLoS ONE | www.plosone.org 11 March 2009 | Volume 4 | Issue 3 | e4854