Novex semy dry blotter

Cells from vinifications were taken and frozen to be broken afterwards with one volume of glass beads in a buffer that contained Tris-HCl 0.1 M, pH 7.5, NaCl 0.5 M, MgCl₂ 0.1 M, NP40 1% (v/v), PMSF 10 mM and protease inhibitors (complete Mini, EDTA-free from Roche) and phosphatase inhibitors^{20 (link)}. Extracts were diluted after quantification by the Bradford method (Biorad Inc. Hercules, CA, USA) in loading buffer for SDS-PAGE. After electrophoresis in an Invitrogen mini-gel device, the gel was blotted onto PVDF membranes for Western blot analysis with a Novex semy dry blotter (Invitrogen, Carlsbad, CA, USA). The anti-phospho-S6 ribosomal protein antibody was obtained from Cell Signalling Technology (Beverly, MA, USA) and the anti-Rps6 antibody was acquired from Abcam (Cambridge, MA, USA). The ECL Western blotting detection system (GE) was used following the manufacturer´s instructions.

Proteins were extracted by a method that implies fast cell lysis with tricholoacetic acid (TCA) [28 (link), 44 (link)]. To 5 OD₆₀₀ units of cells, 5.5% TCA was added and incubated on ice for 15 min before centrifuging cells. The pellet was washed twice with acetone, air-dried and frozen at − 80 °C. Cells were resuspended in 150 μL of 10 mM Tris–HCl, pH 7.5, 1 mM EDTA and broken with 150 μL of 0.2 M NaOH. Cells were centrifuged and the pellet resuspended in gel loading buffer for SDS-PAGE. Alternatively, cells were broken with glass beads and whole cell extracts were extracted in lysis buffer (Tris–HCl 0.1 M, pH 7.5, NaCl 0.5 M, MgCl₂ 0.1 M, NP40 1% (v/v), PMSF 10 mM and protease inhibitors and phosphatase inhibitors) in a FastPrep-24 (MPBio) shaker for three cycles of 20 s at a speed of 4.5 m/s [45 (link)]. Extracts were clarified by centrifugation before being diluted after quantification by the Bradford method (Biorad Inc. Hercules, CA, USA) in loading buffer. Samples were boiled for 5 min before loading. SDS-PAGE was carried out in an Invitrogen mini-gel device. Afterward, the gel was blotted onto PVDF membranes for the immunodetection analysis with a Novex semy dry blotter (Invitrogen, Carlsbad, CA, USA). The used antibodies are described in Additional file 5. The ECL Western blotting detection system (GE) was used following the manufacturer´s instructions.

The cells from the different growth conditions were taken and frozen to be broken afterward with one volume of glass beads in lysis buffer (Tris-HCl 0.1 M, pH 7.5, NaCl 0.5 M, MgCl₂ 0.1 M, NP40 1% (v/v), PMSF 10 mM and protease inhibitors and phosphatase inhibitors) in a FastPrep 24 shaker for three cycles of 20 seconds^{16 (link)}. Extracts were clarified by centrifugation and were diluted after quantification by the Bradford method (Biorad Inc. Hercules, CA, USA) in loading buffer for SDS-PAGE. After electrophoresis in an Invitrogen mini-gel device, the gel was blotted onto PVDF membranes for the Western blot analysis with a Novex semy dry blotter (Invitrogen, Carlsbad, CA, USA). The anti-phospho-S6 ribosomal protein (1:1000 dilution) and the anti-PRX antibodies (1:1000 dilution) were obtained from Cell Signalling Technology (Beverly, MA, USA). The anti-Rps6 antibody (1:1000 dilution) was acquired from Abcam (Cambridge, MA, USA), and anti-GFP (1:1000 dilution) and anti-HA (1:2000 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies HRP-conjugated were purchased from Santa Cruz Biotechnology and used at a dilution of 1:5000. The ECL Western blotting detection system (GE) was used following the manufacturer’s instructions.