Multi beads shocker

Skin or tumor samples were snap-frozen in liquid nitrogen, ground to a fine powder by Multi-Beads Shocker (Yasui Kikai, Inc.), lysed in SDS sample buffer and prepared as previously described [27 (link)]. Protein samples (30 μg) were subjected to SDS-polyacrylamide gel electrophoresis, and immunoblot analysis was performed as previously described [27 (link)]. The antibodies used in this study were: rabbit polyclonal anti-TGF-β1 (V) antibody (Santa Cruz Biotechnology), rabbit monoclonal anti-phospho-Smad2 (Ser465/467) (138D4) antibody (Cell Signaling Technology), rabbit monoclonal anti-Smad2 (D43B4) antibody (Cell Signaling Technology) and mouse monoclonal anti-β-actin (AC-74) antibody (Sigma-Aldrich, Saint Louis, MO). To confirm equal loading, membranes were reprobed with anti-β-actin antibody.

Whole-skin samples were snap-frozen in liquid nitrogen and ground to a fine powder using Multi-Beads Shocker (Yasui Kikai Corporation, Osaka, Japan). Total RNA was isolated from cultured cells or tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA), treated with DNase I (Qiagen, Hilden, Germany) and further purified using an RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. The concentration and purity of the extracted RNA were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE). At least two independent RNA extractions were performed for each sample. Total RNA (500 ng) was then reverse-transcribed into cDNA using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Q-PCR analysis was performed using an MX3005P system (Agilent Technologies, Santa Clara, CA, USA) with THUNDERBIRD SYBR qPCR Mix (Toyobo) following the manufacturer's instructions. All Q-PCR reactions were performed in duplicate and the results were analyzed using the comparative Ct method. The Ct values of samples and controls were normalized to those of Gapdh. All primer sets used in this study are shown in Supplementary Table S1.

TS269 (3FLAG-TOR1) or TS270 (3FLAG-TOR1 KOG1-13myc) cells were cultured in YPD medium to an OD₆₀₀ of 2.0–3.0 before being collected by centrifugation, suspended in lysis buffer A (40 mM HEPES-KOH [pH 7.5], 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.3% CHAPS, 1 mM phenylmethylsulfonyl fluoride, 40 μg mL⁻¹ of aprotinin, 10 µg mL⁻¹ of pepstatin A, 20 µg mL⁻¹ of leupeptin), and disrupted using a Multi-Beads Shocker (Yasui Kikai, Osaka, Japan) with 0.5 mm zirconia beads (Nikkato, Osaka, Japan). After the samples had been centrifuged at 12,000 × g for 15 min at 4 °C, the supernatants were collected, the indicated amount of each amino acid was added alongside purified GST-Pib2, and the mixture was incubated for 1 h on ice. Glutathione Sepharose beads were then added and the mixture was rotated for 1 h at 4 °C. After the beads had been washed three times with lysis buffer A containing each amino acid at the indicated amount, the beads were suspended in Laemmli sample dye and boiled for 4 min. The supernatants were subjected to western blotting and the blotted bands were quantified as described above.