For the analysis of phosphorylation sites of OST1, 8 g pSuper:OST1-Myc plants materials, treated with 4oC for 0 or 2 h, were used for protein extraction and immunoprecipitated with anti-Myc agarose. Then OST1-Myc proteins were eluted with 0.1 M glycine solution (pH 2.8) and mixed with 1 M Tris solution to ensure the pH of OST1-Myc is 7.5. After the concentration of PP2CG1 proteins with 30K-ultrafiltration tubes, phosphatase assay was performed: OST1-Myc was treated with GST-PP2CG1 for 0 or 60 min under 30oC. Then, the samples were digested with trypsin using FASP method. The collected solution containing digested peptide were lyophilized for phospho-peptides enrichment, which was performed using TiO2-tips (Thermo Scientific, #A32992) according to the manufacture’s protocol. The phosphor-peptides were resuspended in 0.1% FA for nano LC-MS analysis.