P rps6

After 24 h of treatment, mesangial cells were lysed with lysis buffer (1 M Tris (pH = 7.4), 1.5 M NaCl, 1% Triton-X, 10% Glycerol, 50 mM EDTA (pH = 8), 0.1 M Sodium vanadate, 0.1 M PMSF, 0.1% protease inhibitor cocktail (Calbiochem) Samples were boiled for 5 minutes, electrophoresed on 8% or 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with specific Antibodies (p-AKT, p-GSK3β, p-FoxO3a, PTEN, p-(rp)S6 and p-mTOR, (Cell Signaling) actin HRP (Sigma). Blots were developed using horseradish peroxidase-conjugated secondary Abs and the ECL detection system (Thermo scientific, Pierce research protein products (Rockford, IL, USA)).

Lung cancer cells or tumor tissues were lysed and sonicated in RIPA buffer supplemented with protease inhibitors (Solarbio, Beijing). Protein extracts were boiled in sample buffer (Solarbio), separated by SDS-PAGE and transferred to nitrocellulose filter membranes (Millipore). After blocking in PBS/Tween-20 containing 5% BSA, the membranes were incubated with the primary antibodies. The following antibodies were used: PCNA (#2586), GAPDH (#5174), p-Erk1/2 (#9101), Erk1/2 (#9102), p-AKT (S473, #9271), AKT (#2920), p-RPS6 (#2215), RPS6 (#2217), p-mTOR (#5536) and mTOR (#2983), purchased from Cell Signaling Technologies; MAL2 from Bioss (bs-7175R). β-Actin (ab179467) was purchased from abcam. Signal visualization was detected with ECL substrate (millipore) and a Biotek imaging system. Immunohistochemistry was performed as previously described [35 (link)].

The cells were washed twice with cold PBS and then harvested for Western blotting. Cells were lysed in cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors for 30 min. The lysates were then centrifuged and the supernatants collected. Approximately 40 μg of total protein was denatured and separated by 10% SDS-PAGE, and then transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 hours at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C: PCNA (Abcam, USA), E-cadherin (Cell Signaling Technology, USA), Vimentin (Santa Cruz Biotechnology, USA), p-RPS-6, RPS-6, p-AKT, AKT, p-ERK1/2, and ERK1/2 (Cell Signaling Technology, USA). β-actin (Santa Cruz Biotechnology, USA) was used as a loading control. The membranes were washed five time with TBST and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The signals were visualized using an Enhanced Chemiluminescence Detection Kit (Pierce Biotechnology, USA).

The following antibodies were used for IHC and Western immunoblotting: ATF4 (catalog 10835-1-AP) and SKP2 (catalog 15010-1-AP) from Proteintech; CHOP (catalog 2895), p-YAP (catalog 13008, S127), YAP (catalog 14074), p27 (catalog 3686), MYC (catalog 9402), p-RPS6 (catalog 4858, S235/236), t-RPS6 (catalog 2217), p-mTORC1 (catalog 5536, S2448), t-mTORC1 (catalog 2983), p-ERK1/2 (catalog 9101, T202/Y204), t-eIF2α (catalog 2103), p-AKT (catalog 9271, S473), t-AKT (catalog 4691), EIF4FBP1 (catalog 2855, T37/46), K48-Ub (catalog 8081), p-PERK (catalog 3179, T980), and PERK (catalog 3192) from Cell Signaling Technology; CYR61 (catalog E-AB-14920), CTGF (E-AB-12339), and aquaporin 2 (E-AB-30540) from Elabscience Biotechnology; CDK1 (catalog SC-54) and t-ERK1/2 (catalog SC-94) from Santa Cruz Biotechnology; α-tubulin (catalog T5168) and K63-Ub (catalog 05-1308) from MilliporeSigma; PCNA (catalog MS-106-P1ABX and, p-LATS1/2 (catalog PA5-64591, S809/S872) from Thermo Fisher Scientific; Oct4 (catalog NB100-2379) from Novus Biologicals; Sox2 (catalog ab97959), vimentin (catalog ab92547), Ki-67 (clone SP6), p-eIF2α (catalog ab32157, S51), BIM (catalog ab32158), and TAZ (catalog ab224239) from Abcam; GRP78 (catalog A0241) from StressMarq Biosciences; and nestin 1 (monoclonal rat-401s) from DSHBU Iowa.

Immunohistochemistry (IHC) for all targets was performed using standard protocols on the automated immunohistochemistry staining system Discovery XT (Roche/Ventana, Tucson, Arizona, USA) and the LEICA BOND-III automated stainer (Leica, Wetzlar, Germany) respectively. The following antibodies were used: PD-L1 (Cell Signaling, Boston, U.S.A.), p-S6K1 (Cell Signaling, Boston, U.S.A.), p-4EBP1 (Cell Signaling, Boston, U.S.A.), p-RPS6 (Ser 235/236 and Ser240/244, Cell Signaling, Boston, U.S.A.), p-PRAS40 (Cell Signaling, Boston, U.S.A.), p-NDRG1 (Cell Signaling, Boston, U.S.A.), p-mTOR (S2448, Cell Signaling, Boston, U.S.A.), IDH1_R132H (Dianova, Eching, Germany) and BRAF V600E (DCS, Hamburg, Germany) [27 (link)].

Liver specimens were fixed in 4% paraformaldehyde overnight at 4°C and embedded in paraffin. Hematoxylin & Eosin (H&E) staining on 4μm liver sections was performed to characterize histopathologically liver preneoplastic and neoplastic lesions in mice. Immunohistochemistry was performed as described previously [29 (link)]. Briefly, antigen retrieval was achieved in deparaffinized sections by boiling in 10mM sodium citrate buffer (pH 6.0) for 10 min. After a blocking step with the 5% goat serum and Avidin-Biotin blocking kit (Vector Laboratories, Burlingame, CA), the slides were incubated with primary antibodies overnight at 4°C. Primary antibodies used for the experiment are as follows: p-RPS6 (4858; Cell Signaling Technology), Ki67 (RM-9106; Thermo Fisher Scientific), p-4EBP1 (2855; Cell Signaling Technology), and SLC38A1 (HPA052272; Sigma-Aldrich, St. Louis, MO). These primary antibodies were selected for the analysis since they have been extensively validated by the manufacturers for immunohistochemistry. After washes, slides were incubated in 3% H2O2 for 20 minutes to quench the endogenous peroxidase, then followed by one hour of secondary antibody incubation. Signal was detected by the Vectastain ABC Elite Kit (Vector Laboratories) and visualized by DAB (Vector Laboratories).