Anti sumo1

For in vitro SUMOylation assays, proteins were incubated in 20 µL of reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 15% glycerol, 5 mM ATP, 10 mM MgCl2, pH 7.8) with or without 1 µg of SUMO1 protein (Abcam, Cambridge, UK), E1 (0.5 µg of human SAE1 full and 0.5 µg of SAE2/UBA2 peptide), E2 (2 µg of MdSCE1 customized by Abmart), and 8 µg of GST-MdSIZ1 or His-MdMYB30 protein for 3 h at 37°C. Next, 10% SDSpolyacrylamide gel immunoblot analysis was performed with anti-His and anti-SUMO1 antibodies (Abcam). For in vivo SUMOylation assays, three 3-week-old transgenic apple calli were extracted with IP lysis/wash buffer, and soluble extracts were IPed with anti-Flag antibody using a Pierce Crosslink IP kit following the manufacturer's instructions. Lastly, 8% SDS-polyacrylamide gel immunoblot analysis was performed with anti-Myc (Abmart), anti-GFP(Abmart), and anti-SUMO1(Abcam) antibodies. For the SUMO conjugate assay, total proteins of plant tissue were extracted with 50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.2% Triton X-100, 1 mM PMSF, and 5 mM EDTA. Next, 10% SDS-polyacrylamide gel immunoblot analysis was performed with anti-SUMO1 and anti-actin antibodies (Abcam). Anti-actin antibody was used to ensure equivalent protein loading.