Secondary anti rabbit and anti goat antibodies

The procedure was performed, as previously described, following a standard immunoblotting protocol [38 (link)]. After incubation, the LNT-229 cells were washed with ice-cold phosphate buffered saline (PBS) and pelleted. The lysates were prepared using a lysis buffer, P, with the addition of 1% Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo Fisher Scientific, Hamburg, Germany), diluted in a Laemmli buffer and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Subsequently, the proteins were blotted to nitrocellulose membranes (0.45 µm; GE Healthcare, Little Chalfont, UK). The membranes were probed with antibodies to phosphorylated (p)-AMPK (Thr172) (1:1000; #2531 Cell Signaling Technology, Danvers, MA, USA), p-ACC (Ser79) (1:1000; #3661 Cell Signaling Technology, Danvers, MA, USA) and actin (#sc-1616, 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary anti-rabbit and anti-goat antibodies were purchased from Santa Cruz Biotechnology. Enhanced chemiluminescence was used for detection [37 (link)].

After the incubation period, cells were washed with ice-cold PBS and harvested into ice-cold PBS containing protease inhibitors. Lysates were prepared as described using lysis buffer P and subjected to SDS-PAGE analysis [17] (link). Membranes were probed with antibodies to HIF-1α (BD Transduction Laboratories # 610958) or actin (Santa Cruz Biotechnology, # sc-1616). The secondary anti-rabbit and anti-goat antibodies were purchased from Santa Cruz Biotechnology. Chemiluminescence solution was used for detection and was composed of 1 ml solution A (200 ml 0.1 M Tris-HCl pH 8.6, 50 mg Luminol), 100 µl solution B (11 mg p-hydroxycurmarinacid, 10 ml DMSO) and 0.3 µl H₂O₂ (30%).

Cells were washed and scraped with ice-cold PBS. Lysates were prepared using lysis buffer P containing protease and phosphatase inhibitors (#1861280; Thermo Scientific, Dreieich, Germany). 10 μg of protein per condition were subjected to SDS-PAGE analysis. Membranes were probed with antibodies against phospho-4EBP1 (Thr37/46) (236B4; Cell Signaling), phospho-RPS6 (Ser 240/244) (D68F8; Cell Signaling), phospho-RPS6 (Ser 235/236) (D57.2.2.E; Cell Signaling), RPS6 (5G10; Cell Signaling), 4EBP1 (53H11; Cell Signaling) or actin (# sc-1616 Santa Cruz Biotechnology, Dallas, Texas, USA). The secondary anti-rabbit and anti-goat antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Chemiluminescence solution was used for detection and was set up with 1 ml solution A (200 ml 0.1 M Tris-HCl pH 8.6, 50 mg Luminol), 100 μl solution B (11 mg p-hydroxycurmarinacid, 10 ml DMSO) and 0.3 μl H₂O₂ (30%).