P70s6k

The expression of target proteins was assessed by western blot. After transfection for 24 h, the cells were starved for 24 hours inmedium with 1% FBS. Then total proteins from gastric cancer cells were extracted by using RIPA buffer with 1% protease inhibitor. Proteins were concentration-measured by BCA method and subjected to SDS-PAGE at 20 μg total protein per lane. Immunodetection was performed with standard techniques. Antibodies to Bcl2, Bax, Active-Caspase-3, AKT, p-AKT, p70S6K, Cyclin D1 and α-Tubulin were purchased from Abcam (Shanghai, China). The visualization of protein bands was performed by using the ECL development system. ImageJ software was used to analyze the protein densities.

BMECs were collected using 0.25% trypsin and lysed in RIPA buffer (Solarbio, China) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF; Pierce, USA) and 1% phosphatase inhibitor cocktail (Roche). Western blotting was performed as previously described [4 (link)], using primary antibodies against β-actin (mAbcam 8226, 1:1,000, Abcam), peroxisome proliferator-activated receptor gamma (PPARγ; EP4394(N), 1:1,000, Abcam), fatty acid synthase (FASN; ab99359, 1:2,000, Abcam), protein kinase B (Akt, #9272, 1:500, CST), pho-Akt (Ser473, 1:500, CST), mTOR (5536 T, 1:1,500, Univ), pho-mTOR (5536 T, 1:1500, Univ), ribosomal protein S6 kinase B1 (P70S6K; 2708 T, 1:1,500, Univ), pho-P70S6K (9234 T, 1:1,500, Univ), Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 (4E-BP1; 9644 T, 1:1500, University), Pho-4E-BP1 (2855 T, 1:1,500, Univ), and RAI14 (EPR8518, 1:1,000, Abcam).

The tissue samples were fixed in 10% neutral formalin and embedded in paraffin for sectioning and staining according to the manufacturer’s instructions. The 3- to 5-μm-thick tissue sections were deparaffinized and hydrated in xylene and serially diluted ethanol, respectively. The endogenous peroxidase was blocked by incubation with 3% H₂O₂ for 10 min. Antigen retrieval was performed in a microwave oven using citrate solution. Subsequently, the tissue sections were incubated with the appropriate primary antibody for 12 h to 16 h at 4°C. The slides were washed three times in phosphate-buffered saline (PBS) and incubated with secondary antibody for 20 min. After three further washes in PBS, diaminobenzidine tetrahydrochloride (DAB) was used before counter staining with hematoxylin. The following primary antibodies were used: mTOR (1:150), p-mTOR (1:200) (Abcam, Cambridge, UK), p70S6K (1:150) (Abcam, Cambridge, UK) and p-p70S6K (1:100) (Elabscience, Wuhan, China).

Cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche) for 30 min and then centrifuged to remove the Triton-insoluble pellet. Samples normalized with equal protein concentration were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. Polyvinylidene fluoride (PVDF) membranes were incubated with the following primary antibodies for overnight at 4°C: UBQLN1 (Abcam, 1:2000), p-mTOR (Abcam, 1:2000), mTOR (Abcam, 1:2000), p-p70S6K (Abcam, 1:2000), p70S6K (Abcam, 1:2000), GAPDH (Sigma, 1:1000). Goat anti-mouse (1:5000) or anti-rabbit (1:5000) secondary antibodies (BBI Life Science Corp.) were used. The protein bands were visualized using chemiluminescence imaging system V1.8.0.112 (Tannon Science & Technology Co., Ltd,). All experiments were independently performed in triplicates [19 ].

The protein concentration was measured with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, IL). Aliquots containing 20 mg protein samples were separated by 8%-12% SDS-polyacrylamide gel. Then, the membranes were immunoblotted separately with primary antibody against DGAT2 (1 : 1000, Novus Biologicals), p-HSL (1 : 1000, Cell Signaling Technology), ATGL (1 : 1000, Bio-Techne), MTP (1 : 1000, Absin), LC3A/B (1 : 1000, Cell Signaling Technology), P62 (1 : 1000, Cell Signaling Technology), mTOR (1 : 1000, Cell Signaling Technology), p-mTOR (1 : 1000, Cell Signaling Technology), p70S6K (1 : 1000, Abcam), p-p70S6K (1 : 1000, Abcam), and GAPDH (1 : 2000, Proteintech) antibodies at 4°C for overnight. Protein bands were assessed using ECL solution (Beyotime, China) and detected by Image Quant LAS-4000 mini (GE, USA). The band intensity was assessed with GELPRO32.

The following antibodies were used for immunofluorescence14 and Western blot experiments: LAMP1 (Sigma; #SAB3500285), LC3 (Abcam; #ab62721), β‐Actin (Sigma; #A5441), cathepsin D (SANTA CRUZ; # SC‐377299), Cathepsin L (Abcam; #ab6314), GAPDH (Abcam; #ab9484), TFEB (Sigma; # 3110428), Histone H3 (Cell Signaling; #4499), mTOR1 (Cell Signaling; #2971), p‐mTOR1 (Cell Signaling; #5536), P70S6K (Abcam; #ab32529), p‐P70S6K (Cell Signaling; #9206), AMPK (Cell Signaling; #5831), p‐AMPK (Cell Signaling; #2535), ACC (Cell Signaling; #3676), p‐ACC (Cell Signaling; #11818), ULK1 (Cell Signaling; #8054), p‐ULK1 (Cell Signaling; 5869), DAPI (Sigma; #D9564), Fluoromount™ Aqueous Mounting Medium (Sigma; #SLBN4103V), EX527 (Aladdin Industrial Corporation; #E129892), Resveratrol (Aladdin Industrial Corporation; #R107315).