Cocktail inhibitor

The total protein extract of leaves from Juniperus oxycedrus subsp. oxycedrus, Juniperus oxycedrus subsp. badia and Corema album were obtained by Jacobs method [35 (link)]. The leaves were powdered under liquid nitrogen containing polyvinylpolypyrrolidone (0.5 g PVPP per g of fresh weight), were add 10 mL·per 1 g fresh weight, at 4 °C, of Tris-HCl 350 mM, pH 8.0, containing 11 mM sodium diethylcarbamate and 15 mM cysteine. A protease inhibitor cocktail for complete inhibition of serine and cysteine proteases (Complete, EDTA free inhibitor from Roche Switzerland) was added at the beginning of the extraction procedure (1 tablet of cocktail inhibitor for each 10 mL of extraction buffer). The slurry was centrifuged at 18,000× g for 15 min at 4 °C (Beckman J2–21M/E, rotor JA 20000), and the supernatant desalted through PD-10 columns (GE Healthcare), with a Sephadex G-25 Medium previously equilibrated in 50 mM Tris–HCl buffer (pH 7.5) containing 2 mM CaCl₂ and 2 mM MgCl₂, and dialyzed against saline containing 2 mM CaCl₂ and 2 mM MgCl₂.
The total protein extract from Arbutus unedo L. were obtained by an optimized method of extraction with buffer containing 5% glycerol and 1% Triton X-100, described by Silva and Souza [36 (link)].

Protein samples from the indicated cells were prepared in a RIPA buffer (Beyotime, Shanghai, China, P0013C) with a cocktail inhibitor (Roche, Germany, 11836170001) and boiled for 5 min. 20 μg of each sample was loaded into SDS-polyacrylamide gel. After SDS-PAGE, the proteins in the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Germany, IPVH00010). The membrane was then blocked in 5% fat-free milk for 2 h at room temperature and then incubated with primary antibodies overnight at 4 °C. The corresponding secondary antibodies were incubated for 1 h before protein detection using an Odyssey Imaging System (Li-COR, Lincoln, NE, USA).

Cells were ruptured with RIPA buffer (Beyotime) containing cocktail inhibitor (Roche). Cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore). The membranes were blocked and then incubated with primary antibodies overnight at 4 °C. Specific antibodies used are listed below: METTL3 (Cell Signaling Technology), YTHDF2 (Cell Signaling Technology) and GAPDH (Proteintech). Subsequently, the membranes were incubated with corresponding secondary antibodies and detected by ECL Western Blotting Substrate (Thermo).