Protein extraction and Western blot
The total proteins were extracted from apple tissues, including apple calli and leaves, using the protein extraction solution: 100 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid disodium salt, 1% polyvinylpyrrolidone K30, 0.07% β-mercaptoethanol, and 6.85% sucrose. Prepared protein samples were separated on a 10% gel using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche). Then specific antibodies were applied to detect corresponding proteins, and peroxidase-conjugated secondary antibodies (Adcam) were utilized to visualize the immunoreactive proteins through an ECL detection kit (Millipore). MdACTIN was used as loading control.