Anti flag beads

Cells were collected 24 h after transfection and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1 mM EDTA, 1% NP-40, and 0.5% SDS supplemented with protease inhibitor from Roche). Whole cell lysates were precleared with protein A/G beads (Abmart) and incubated with anti-FLAG beads or anti-MYC beads (Abmart) overnight at 4°C. After extensive washing, the beads were boiled in sample loading buffer for 10 min to elute precipitated proteins for immunoblots.
Transfected cells were likewise harvested for immunoblot analyses. Protein lysates or immunoprecipitates were separated on SDS-PAGE gels by electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk (BD-Pharmingen) and incubated with antibodies for proteins of interest. Blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) according to the manufacturer’s instructions.

For protein extraction, the mycelia of different strains cultured in liquid CM for 48 h were collected, which were ground into powder in liquid nitrogen and resuspended in protein extraction buffer (Biyuntian, Beijing, China). To detect O-GlcNAcylation level, the total protein was immunoprecipitated using anti-Flag beads (Abmart, Shanghai, China), which was then separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany). The PVDF membrane was then incubated with anti-O-GlcNAc as the primary antibody (1:5000, Sigma-Aldrich, St. Louis, MO, USA) and anti-rabbit horseradish peroxidase as the secondary antibody (1:10,000, Abmart, Shanghai, China). To detect protein level of Def1, the total protein was separated on a 10% SDS-PAGE gel and, then, transferred onto a PVDF membrane, which was incubated with anti-Flag as the primary antibody (1:5000, Abmart, Shanghai, China) and anti-rabbit horseradish peroxidase as the secondary antibody (1:10,000, Abmart, Shanghai, China).

Ten-day-old pPXL1:PXL1-FLAG transgenic seedlings were tested to observe the phosphorylation status of PXL1. The seedlings were cultured in 1/2 MS liquid medium treated with mock or 20 µM CLE19, CLE19_G6T or CLV3 peptide for 1.5 h and then ground to a fine powder in liquid N₂ for protein extraction performed as described above. The extracted protein was analyzed by Phos-tag (Wako) gel and immunoblotting using anti-FLAG (GNI) antibody. The anti-HSP70 (Abmart) antibody was used to determine the amount of input protein. anti-FLAG beads (Abmart) were used for immunoprecipitation to purify the PXL1-FLAG protein. Anti-pS/T (ECM) was used to detect the phosphorylation status of the PXL1 protein after CLE19 treatment. The seedling protein extracted was also treated with λpp (NEB) at 30 °C for 0.5 h and then analyzed by anti-FLAG and anti-HSP antibodies.