Anti p70s6k

Protein samples from cultured cells were prepared by direct lysis in 5× SDS sample buffer, followed by heating at 95 °C for 5 min. Samples were resolved on 8–12% SDS–polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5% (w/v) dried milk in Tris-buffered saline/Tween (TBST) for 1 h at room temperature, after which they were incubated with primary antibody, followed by the correct horseradish-peroxidase-conjugated secondary antibody. Blots were developed using Western Bright ECL substrate (Advansta) or Immobilon Western chemiluminescent substrate (Millipore) using a Bio-Rad Gel Doc. Antibodies used were anti-HIF-1α (catalogue no. 14179), anti-IκB-α (9242), anti-phospho-NF-κB-p65 (3033), anti-NF-κB-p65 (8242), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (catalogue no. 9212), anti-phospho-ERK (9101), anti-ERK (4695), anti-phospho-p70-S6K (catalogue no. 9205), anti-p70-S6k (9202), all purchased from Cell Signaling Technology, anti-IL-1β (R&D Systems, AF401-NA), anti-β-actin (Sigma-Aldrich, AC-74), anti-FLAG (Sigma-Aldrich, F1804), L-2HGDH (Antibody Genie, CAB7996), and D-2HGDH (Antibody Genie, CAB16213). anti-HIF-1α from Novus (catalogue no. NB100-449) was used for human blots. Secondary antibodies used were horseradish-peroxidase-conjugated anti-mouse IgG, anti-goat IgG and anti-rabbit IgG (Jackson ImmunoResearch).