Phospho s6 ribosomal protein ser235 236

N-Hydroxyphthalimide was purchased from Accela ChemBio Co., Ltd. (Shanghai, China). Propidium iodide (PI), RNase A and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich. Antibodies of mTOR, Phospho-mTOR (Ser2448), Phospho-mTOR (Ser2481), S6K1, Phospho-S6K1 (Thr389), Phospho-S6 Ribosomal Protein (Ser235/236), 4E-BP1, Phospho-4E-BP1 (Ser65), Phospho-Akt (Ser473), Phospho-Akt (Thr308), Cleaved PARP, Cleaved Caspase 3, Caspase 9, cyclin B1, cdc2 were obtained from Cell Signaling Technology; antibodies of S6, Akt, P-ERK1/2, β-actin, Caspase 3, Caspase 8, Bcl-xL, survivin were from Santa Cruz; antibody of ERK was from Epitomics; antibody of eIF4E was obtained from BD Biosciences; Alexa Fluor® 647 donkey anti-mouse IgG antibody was purchased from Invitrogen; all the other secondary antibodies were from Sigma-Aldrich.

The antibodies used in the present study were purchased from Cell Signaling Technology, including Bcl-2, Bax, caspase 3, β-actin, LC3, AKT, phospho-AKT (Ser473), mTOR, phospho-mTOR (Ser2448), S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236) and ATG5, with their catalog number D17C4, D2E11, D3R6Y, E4D9Z, D11, 11E7, D9E, 7C10, D9C2, 54D2, D57.2.2E, and D5F5U, respectively. Other antibodies included cytochrome c (M1701-9), goat anti-rabbit/mouse IgG-HRP (ImmunoResearch Laboratories) and FITC goat anti-mouse IgG (Beyotime, A0562).
Reagents used in our research included: cell cycle and cell apoptosis analysis kits (KEYGEN, KGA511 and KGA107); Hochest33342 staining kit (Beyotime, C1025); mitochondrial membrane potential detection kit and ROS measurement kit (NJJCBIO, G009-1-3 and E004-1-1), MitoTracker Deep Red FM (Life Technologies, M22426), Lyso-Tracker Red (Invitrogen, L7528), cholorquine (CQ, PubChem, 2719), 3-MA, and N-acetylcysteine (NAC) (aladdin, M129496 and A105422). Moracin N (MAN) was isolated and purified by Prof. Tian Jingkui from the Key Laboratory of Biomedical Engineering at Zhejiang University.

For histological analyses, mouse uteri were fixed in 10% formalin solution (Sigma, MO, USA) overnight at 4°C and then transferred to 70% ethanol until processing. The fixed tissues were dehydrated in a series of graded ethanol, cleared in xylene, and embedded in paraffin wax. Embedded tissue samples were sectioned at 6µm and mounted on slides. Haematoxylin and eosin (H&E) staining and IHCs were performed using standard protocols described earlier [23 (link)]. Tissue sections were incubated overnight at 4°C with following primary antibodies: Phospho-S6 Ribosomal protein Ser235/236 (1:400; Cell Signalling Technologies, MA, USA), CK8 (Developmental Studies Hybridoma Bank, IA, USA). Biotinylated secondary antibodies (Jackson ImmunoResearch Labs, PA, USA or Thermo Fischer Scientific, Australia) were used followed by incubation with horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific). Sections were then exposed to Diaminobenzidine (DAB, Sigma) to develop colour. Sections were counterstained with hematoxylin. Images were captured using Olympus DP72 microscope and the Aperio AT2 slide scanner (Leica Biosystems, Victoria, Australia). The gain and exposure time were set constant across tissue samples.

The stomachs taken from mice were opened in the minor curvature, fixed in 4% formaldehyde for 2 h at room temperature, dehydrated and embedded in paraffin wax. 2–3 μm sections were deparaffinized in xylene, and rehydrated in a graded series of ethanol. Antigen retrieval was performed when necessary in boiling natrium citrate buffer (PH6.0) for 15 min. Antibodies directed at Glucagon (Cell signaling,1:100), Chromogranin B (Santa Cruz, 1:100), SV40 T Ag (Santa Cruz, 1:500), Ki-67 (Dako, 1:50), Hydrogen Potassium ATPase Beta (Abcam, 1:1000), Phospho-S6 Ribosomal Protein (Ser235/236) (Cell signaling, 1:100), Phospho-S6 Ribosomal Protein (Ser240/244) (Cell signaling, 1:800), were applied to the sections for 2 h incubation at room temperature. Sections were then treated with HRP-coupled secondary antibodies (ImmPRESS Anti-Rabbit Ig Polymer Detection Kit, Vector Labs) or biotin-conjugated secondary antibody followed by avidin-biotin-peroxidase complex (VECTASTAIN Elite ABC Kit, Vector Labs). Primary antibodies were visualized by AEC or DAB as substrate.