Hpa021241

Tumor pieces were homogenized in 1 mL RIPA buffer [25 mM Tris-Cl, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 1x cOmplete protease inhibitor (Roche, Basel, Switzerland)] using a GentleMACS tissue homogenizer (Miltenyi Biotec, Bergisch Gladbach, Germany). For tissue culture cells, cells were scraped in 300 μL RIPA buffer. In each case, the resulting lysate was clarified by centrifugation at 21000 × g for 20 min. Protein concentration of the lysate was determined by BCA assay (Thermofisher). Lysates were resuspended at 1 mg/mL in Laemmli SDS-PAGE sample loading buffer (10% glycerol, 2% SDS, 60 mM Tris-Cl pH 6.8, 1% b-mercaptoethanol, 0.01% bromophenol blue) and denatured at 100°C for 5 min. Extracts (30 μg of protein) were resolved by SDS-PAGE using 12% acrylamide gels running at 120 V until the dye front left the gel. After SDS-PAGE resolution, protein extracts were transferred to nitrocellulose using an iBlot semi-dry transfer system (Thermofisher). Membranes were blocked in 5% non-fat dry milk, incubated in primary antibodies to PHGDH (Sigma-Aldrich, St. Louis, MO, HPA021241, 1:1000) or vinculin (Abcam, Cambridge, MA, ab18058, 1:250) and detected using HRP-conjugated secondary antibodies and chemiluminescence.