Centrivap console

Volumes of 120 μL frozen serum aliquots were mixed with 480 μL extraction solution by mixing methanol and acetonitrile (2:1). The extraction solution contained two standards (2-chloro-L-phenylalanine and decanoic acid). Samples were vortexed for 120 s and then placed at 4°C for 30 min. Following centrifugation for 10 min at 14000 rpm, the supernatant was split into two aliquots (250 μL each aliquot), and one of the supernatant aliquots was used for analysis and one for backup. The aliquot to be analyzed was dried via evaporation in Labconco Centrivap Console and was then dissolved in 125 μL 50% methanol and centrifuged at 14000 rpm for 10 min. The supernatant was moved to 200 μL MicroSert Insert for analysis.
Brain tissue (100 mg) was homogenized in 1000 μL extraction solution by mixing methanol acetonitrile (2:1), then placed at 4°C for 30 min. The extraction solution contained two standards (2-chloro-L-phenylalanine and decanoic acid). Following centrifugation for 10 min at 14000 rpm, the supernatant was split into two aliquots (400 μL each aliquot), one of the supernatant aliquots was used for analysis and the other was stored for backup. The aliquot used for analysis was dried via evaporation in Labconco Centrivap Console and then was dissolved in 200 μL 50% methanol and centrifuged at 14000 rpm for 10 min. The supernatant was moved to 200 μL MicroSert Insert for analysis.

The shoots with concurrent bud break were selected, and the fourth leaf from each shoot was harvested. Twenty leaves were randomly selected and pooled together as one biological replicate; four biological replicates were used. EWs were removed by gum arabic; the film was collected into a glass tube containing 21 ml of chloroform:water (2:1, v/v), and 75 μg of n-tetracosane (Sigma-Aldrich, St. Louis, United States) was added as internal standard. After vigorous agitation and phase separation, the organic phase was transferred into a new glass tube. Extraction was repeated with another 4.5 ml of extraction buffer. The organic phases were combined and evaporated under CentriVap Console (Labconco, KS, United States). The adaxial EWs were firstly removed and then abaxial EWs were isolated from the same batch of leaves.
After EW removal from both leaf surfaces by gum arabic, the leaves were still physically intact, and were used to extract IWs. The adaxial IWs were rinsed five times by chloroform; the elution was collected into a glass beaker. Abaxial IWs were isolated in the same manner. The elution was dried down by CentriVap Console to recover IWs.

The cysteine side chains of pVIc, 8-Actin-C, 11-Actin-C were labelled at a concentration of 200 μM in 25 mM HEPES (pH 7.0), 25 mM NaCl, and 20 mM ethanol by the addition of Cy3B-maleimide to 600 μM. Labelling reactions were incubated at 21 °C in the dark for 2.5 h. Dye-conjugated peptides were purified from unreacted dye and peptide on a 15 cm × 4.6 mm Discovery C18, 5 μm column. Peptides were eluted via a linear acetonitrile gradient from 0 to 40% in 0.1% trifluoroacetic acid. The conjugated peptide peak was identified by MALDI-TOF analysis. The fractions were evaporated to dryness on a LABCONCO Centrivap Console and resuspended in water. The concentration of the dye-conjugated peptide was determined by measuring the dye concentration spectrophotometrically using . The conjugated peptide was aliquoted, dried and stored at –20 °C.