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.