Enhanced chemiluminescence detection

A standard protocol was used for immunoblot analysis [51 (link)]. The commercial antibodies used in this study are listed in Supplementary Table 2. For chemiluminescent detection, blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Cayman, Ann Arbor, MI, USA) at room temperature, followed by enhanced chemiluminescence detection according to the manufacturer's protocol (Millipore, Billerica, MA, USA). All blots were also immunoblotted for β-actin (Sigma, St. Louis, MO, USA) to demonstrate equal loading of protein samples. All immunoblotting experiments were repeated at least 3 times.

Protein samples diluted in RIPA buffer were homogenized with Precellys (Peqlab, Erlangen, Germany), incubated 30 minutes on ice and afterwards centrifuged at 4°C for 20 minutes. The supernatant was used to measure the protein concentration according to Bradford. Western blot analyses were performed with 25 μg protein. After blotting, the nylon membranes were stained with Ponceau S. Molecular weights were determined by comparison with PageRuler prestained molecular weight marker (Fermentas, St. Leon-Rot, Germany). The immunoreactive signals were visualized by enhanced chemiluminescence detection (Millipore, Schwalbach, Germany) and quantified by Fusion Fx7 Imaging System (Peqlab, Erlangen, Germany). The primary antibody against BCAT2 (mouse polyclonal) was purchased from Abcam (ab72850) and diluted 1:1000. An anti-phospho-mTOR (Ser2448) (rabbit Ab, 1:500, #2971) and anti-mTOR (rabbit Ab, 1:500, #2972) antibody (both NEB, Frankfurt, Germany) were used to calculate phosphorylation as ratio of band intensities (p-mTOR vs. mTOR) in the same blot. Protein amount was evaluated by re-blotting with an antibody against the mouse monoclonal beta-actin antibody (Sigma, A-5441, 1:40000) after stripping the membranes. Protein amounts were calculated as ratio of band intensities (BCAT2 vs. beta-actin) in the same blot.

Cell lysates were extracted in RIPA buffer (Pierce) and quantified by a BCA protein assay kit (Pierce) according to the manufacturer's protocol. Equal amounts (30 μg) of total protein were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotted onto activated polyvinylidene difluoride membranes (Millipore). After blocking with 5% fat-free milk, the membranes were incubated with primary antibodies, as listed in Supplementary Table S2. The blots were then incubated with secondary antibody conjugated with horseradish peroxidase and immunoreacted bands were detected by enhanced chemiluminescence detection (Millipore).

Western blotting and immunoprecipitation were performed as previously described [32 (link)]. Briefly, cell lysates were prepared using lysis buffer containing Tris pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 10 nM calyculin A, 1 mM Na₃VO₄ and protease inhibitors, and normalized by protein concentrations using the Bradford method (Bio-Rad). For western blotting, cell lysates were boiled in Laemmli sample buffer and separated on 8%–12% SDS-PAGE and transferred to Immobilon-P PVDF Membrane (Sigma-Aldrich). The membranes were blocked in TBST containing 5% nonfat milk, incubated with primary antibodies based on the manufacturer’s instructions, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Thermo Fisher) and enhanced chemiluminescence detection (Sigma-Aldrich).
For co-immunoprecipitation, cell lysates were incubated with primary antibodies, anti-FLAG M2 affinity agarose gel, or anit-HSP90 conjugated agarose at 4 °C overnight, followed by incubation with protein A/G Sepharose for an additional 1 h at 4 °C, when applicable. Beads were washed three times with lysis buffer and boiled in Laemmli sample buffer, and immune complexes were analyzed by SDS-PAGE and western blotting.