To determine the effects of different light intensities on the stability of the MdTCP46 and MdBT2 proteins, fruit of ‘Red Delicious’ (120 d after full bloom) were subjected to the low- and high-light treatments for 3 d. The peels were collected, and total proteins were extracted and incubated with the MdTCP46-GST and MdBT2-HIS fusion proteins. Samples were taken at 0–6 h of incubation and protein residues were determined using HIS and GST antibodies (Abmart).
To determine the effects of MdBT2 on the stability of the MdTCP46 protein, WT and transgenic apple calli extracts were incubated with the MdTCP46-GST protein. Samples were taken at 0–6 h of incubation and protein residues were determined using a GST antibody (Abmart). Protein degradation assays in vitro were performed as previously described (An et al., 2019b (link), 2020 (link)). Briefly, calli were ground in liquid nitrogen, soaked with protein extraction liquid, and the supernatant was obtained following centrifugation.
To examine the effects of the MG132 proteasome inhibitor, total proteins from calli were pre-treated with 100 µM MG132 dissolved in DMSO for 0.5 h. DMSO alone was used as the blank control.
To determine the effects of MdBT2 on the stability of the MdTCP46 protein, WT and transgenic apple calli extracts were incubated with the MdTCP46-GST protein. Samples were taken at 0–6 h of incubation and protein residues were determined using a GST antibody (Abmart). Protein degradation assays in vitro were performed as previously described (An et al., 2019b (link), 2020 (link)). Briefly, calli were ground in liquid nitrogen, soaked with protein extraction liquid, and the supernatant was obtained following centrifugation.
To examine the effects of the MG132 proteasome inhibitor, total proteins from calli were pre-treated with 100 µM MG132 dissolved in DMSO for 0.5 h. DMSO alone was used as the blank control.