Protease inhibitor cocktail for general use

For normal one-dimensional gel electrophoresis, harvested yeast cells were resuspended in lysis buffer [50 mM Tris-HCl (pH7.6), 0.5 mM EDTA, 150 mM NaCl, 50 mM sodium fluoride, 30 mM β-glycerophosphate, 1 mM PMSF, protease inhibitor cocktail for general use (Nacalai Tesque), and 0.5% Triton X-100]. The cell suspension was continuously vortexed for 10 min at 4 °C in a microtube mixer (MT-360, Tomy) with a half-volume of glass beads to lyse the yeast cells. Unbroken cells and debris were removed by centrifugation at 500 × g for 10 min, and the supernatant fraction containing total 5 μg of protein was used for SDS-PAGE analysis. After separation by SDS-PAGE, proteins were transferred to nitrocellulose membranes and proteins were detected using antibodies. To detect bound antibodies, chemiluminescent substrates Chemilumi-One (Nacalai Tesque) or SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) were used in conjunction with LAS-3000/4000 imager (Fujifilm, Tokyo, Japan).
For co-immunoprecipitation, cell lysates were pre-cleared by 1 hr incubation with Protein G Sepharose 4B or IgG Sepharose. Pre-cleared lysate was subsequently incubated with anti-Ypk1, anti-CBP or anti-HA polyclonal Abs. Washed protein-bound beads were eluted by boiling and analyzed by above-mentioned procedures.

To make protein extracts, frozen livers were weighed and defrosted on ice, then homogenized (g of tissue/20 mL) using an ice‐cold Physcotron homogenizer (Microtec Co., Ltd., Chiba, Japan) in ice‐cold RIPA lysis buffer: 50 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% NP‐40, 1 mmol/L phenylmethylsulfonyl fluoride, and a Protease Inhibitor Cocktail for General Use (Nacalai Tesque, Kyoto, Japan). All protease inhibitors were added immediately prior to use. Crude lysates were incubated at 4ºC for 30 minutes with gentle rotation, before clarification by centrifugation at 20 000× g at 4°C for 30 minutes. The resulting supernatant was carefully collected in a new tube, and the protein concentration was determined by using the BCA Protein Assay Kit (Pierce, Rockford, IL) with BSA as a standard according to the manufacturer's protocol. The liver lysate samples were subjected to immunoblot analyses.

To compare the level of ATML1, ATML1^W471L, PDF2 or PDF2^W463L protein expressed under the constitutive CaMV promoter in the Nicotiana benthamiana leaves (i.e., transient overexpression assay), the appropriate plasmids were introduced into Agrobacterium tumefaciens strain GV3101, and the resulting strains were used for agroinfiltration of Nicotiana benthamiana leaves. The empty plasmid (pRI201AN vector (TaKaRa)) was used as a negative control. Samples were collected on day 2 after infiltration, flash frozen in liquid nitrogen, and stored at − 80 °C prior to protein extraction. Protein extractions were performed as previously described (Mukherjee et al. 2022 (link)). Briefly, frozen samples were homogenized in liquid nitrogen, and hot sodium dodecyl sulfate (SDS) buffer (8 M urea, 2% SDS, 0.1 M DTT, 20% glycerol, 0.1 M Tris–HCl pH 6.8, 0.004% bromophenol blue, and 5 × Protease Inhibitor Cocktail for General Use (Nacalai tesque)) was added prior to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting. Proteins were detected via anti-GFP primary antibody [MBL (598MS); 1:2000] and anti-rabbit IgG HRP-conjugate secondary antibody [Promega (W4018); 1:5000]. Detection of secondary antibodies were performed with the Ez West Lumi One (ATTO) using the Image Quant LAS 4000 mini (GE Healthcare).