Phospho rps6 ser 235 236 d57.2.2 e

Immunoblot was performed as described previously [14 (link)]. 10 μg of protein per condition were used for SDS-PAGE analysis. Membranes were incubated with antibodies against phospho-RPS6 (Ser 240/244) (D68F8; Cell Signaling), phospho-RPS6 (Ser 235/236) (D57.2.2.E; Cell Signaling), phospho-PRAS40 (Thr246) (C77D7, Cell signaling) or actin (# sc-1616 Santa Cruz Biotechnology, Dallas, Texas, USA). The secondary HRP-conjugated antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). A chemiluminescence solution was used for detection [50 (link)].

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%).