Bradford assay reagent

Cells (E. coli, BL21) were induced by 0.1 mM IPTG and allowed to grow for 12 h at 16 °C. MdY3IP1-GST protein was eluted from glutathione–agarose beads. The total proteins of the transgenic apple leaf protoplasts were subsequently extracted in degradation buffer containing 25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl₂, 4 mM PMSF, 5 mM DTT and 10 mM ATP as previously described by Zhao et al. (2016 (link)). The supernatant was collected, and the protein concentration was determined by the using the Bradford assay reagent (Bio-Rad, Hercules, CA, USA). Each reaction mix contained 100 ng of MdY3IP1-GST protein and 500 μg of total protein from WT, 35S::MdDEP1 and TRV-MdDEP1 apple leaf protoplasts. The reaction mixes were incubated at 22 °C, and were stopped by the addition of SDS-PAGE sample buffer and boiled for 10 min. The results were quantified using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA, USA).

For the measurement of the sugar content, total protein content, amino acid composition and energy-related parameters tubers of 30-50 g FW, were harvested from senescent plants, and used. The uber samples were obtained as described in Baroja-Fernández et al. (2009) (link). Three samples were taken from each tuber, immediately freeze-clamped and finally ground to a fine powder in liquid nitrogen.
Soluble sugars (glucose, fructose and sucrose) were determined by HPLC on an ICS-3000 Dionex system as described by Baroja-Fernández et al. (2003) (link). The protein content was determined by using Bradford assay reagent (Bio-Rad XL-100). The starch content was measured enzymatically with an amyloglucosidase-based UV-test Kit (R-Biopharm).
The amylose content in tuber starch was determined using the iodine-based colorimetric method (Andersson et al., 2006 (link)). Adenine nucleotides were extracted using HCLO₄ and measured by HPLC with a Partisil 10-SAX column according to Bahaji et al. (2015) (link). The measurement of reduced (NADH, NADPH) and oxidized nicotinamide adenine dinucleotides (NAD⁺ and NADP⁺) were measured as described by Queval and Noctor (2007) (link). The amino acids profile was analyzed using HLPC, as described in Loiret et al. (2009) (link).

Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay reagent (Bio-Rad) according to manufacturer instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-p21 (1:500; sc-397), anti-IκBα (1:500; sc-371), anti-p53 (1:500; sc-126, all from Santa Cruz Biotechnology), anti-p-p53 (1:500; 9286, Cell Signaling Technology), anti-ID1 (1:2,000; BCH-1-195-14, Biocheck, Foster City, CA, USA), anti-ID2 (1:500, sc-489, Santa Cruz Biotechnology), anti-ID3 (1:500, sc-490, Santa Cruz Biotechnology), anti-ID4 (1:200; ab49261, Abcam), and anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with a horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).

d18:1/16:0 (860516), d18:1/17:0 (860517), d18:1-d₇/15:0 (860681), d18:1-d₇/18:0 (860677) and d18:1-d₇/24:1 (860679) ceramides were purchased from Avanti Polar Lipids. Water (4218-03) was from J.T. Baker. Methanol (MX0486-1) and isopropanol (PX1834-1) were from Sigma-Aldrich. Ammonium formate (A1190) was from Spectrum. Formic acid (28905) was from ThermoFisher Scientific. Guard cartridge (6956) and analytical column (84410) for LC–MS were from Dikma. Cas9 nuclease (M0386M) was from New England Biolabs. Proteinase K (03115828001) was from Roche. PCR reagents were from New England Biolabs and Promega. Reagents for cloning were from Promega and ThermoFisher Scientific. DNA and RNA kits were from Qiagen and ThermoFisher Scientific. Bradford assay reagent was from Bio-Rad. Glassware for lipid extraction was from VWR. Oligos were synthesised at the Massachusetts General Hospital Center for Computational & Integrative Biology DNA Core, the University of Utah DNA Sequencing Core Facility, or IDT.