Itraq reagents kit

For knockdown of FZD6 and GSK-3β, shRNAs against FZD6 and GSK-3β were cloned into PLKO.1 vector and the recombinant constructions were identified by Sanger DNA sequencing. TOP-FLASH luciferase reporter plasmid that contains two sets of 3 copies of the wild-type TCF binding regions was used for monitoring the transcriptional activity of β-catenin and FOP-FLASH with mutated TCF binding region was used as negative control. Primers, shRNAs and antibodies used in this study were listed in Supplementary Tables S4, S5 and S6. Luteolin, Polyethylenimine (PEI), Acetonitrile (ACN, HPLC-grade), formic acid (HPLC-grade), water (HPLC-grade), sodium dodecyl sulfate (SDS), Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and Iodoacetamide (IAA) were purchased from Sigma-Aldrich. Bio-Rad DC protein assay reagents and bovine serum albumin standard were obtained from Bio-Rad Laboratories. iTRAQ reagents kit and trypsin were purchased from AB SCIEX. 30 kDa cut-off filter membrane was from Pall Corporation. Tri-HCl and ammonium bicarbonate were from FASP kit. UltraMicroSpin column was bought from The Nest Group. Other reagents were purchased from Sigma-Aldrich.

For each sample, 100 μg of protein was denatured by adding half the corresponding volume of 2% SDS and reducing with an equal volume of 50 mM Tris-(2-carboxyethyl)-phosphine. Methyl methanethiosulfonate was used to block cysteine residues, as described in the iTRAQ Reagents kit (AB Sciex). For the key developmental stages of both Vitis species, sequencing-grade trypsin was used to digest the proteins at 37 °C for 16 h and the resulting peptides were labeled following the instructions for the iTRAQ reagent kit (Fig. 1). Three biological replicates were analyzed. After labeling, the eight samples were mixed.