Anti p s6k1

Immunohistochemistry staining was performed as previously described^{45 (link)}. The following primary antibodies (anti-METTL1, Proteintech, 1:2000 dilution; Anti-WDR4, Abcam, 1:1000 dilution; Anti-Ki67, Proteintech, 1:8000 dilution; Anti-RPTOR, Proteintech, 1:500 dilution; Anti-pS6K1, Cell signaling technology, 1:1000 dilution; Anti-pULK1, Affinity, 1:1000 dilution; Anti-p4EBP1, Cell signaling technology, 1:1000 dilution) were used for detection of indicated protein expression. IHC staining was evaluated as H-score using QuPath (v0.2.3) by recording the staining intensity and the proportion of the cells with positive staining^{46 (link)}. Tissues with H-score of ≧100 were defined as high expression samples, and tissues with H-score below 100 were defined as low expression samples. For survival analysis, patients were stratified into four groups based on their relative levels of H-scores: (>75%, 50–75%, 25–50%, and 0–25%).

We purchased anti-microtubule-associated protein LC3, P62, CD63, and BECN1 (beclin1, an autophagosome initiator) antibodies from R&D Systems (Minneapolis, MI, United States). Cell Signaling Technology (Danvers, MA, United States) provided the following antibodies: anti-EZH2 (#5246), anti-mTOR (#2983), anti-p-mTOR (#5536), anti-S6K1 (#2708), anti-p-S6K1 (#9204), anti-TSG101 (#28405), and anti-Ki67 (#9449). GSK126 (EZH2inhibitor) (10 μM), PQR620 (50 nM) and bafilomycin A1 (Baf A1, an autophagosome-lysosome fusion inhibitor) (100 nM) were purchased from MedChem Express (Monmouth Junction, NJ, United States). EZH2 plasmids were purchased from Shanghai Genechem Co., Ltd.

Whole cell lysates were prepared as described [11 (link)]. Soluble proteins were analyzed by immunoblotting with anti-MUC1-C (Ab5; Thermo Scientific), anti-TIGAR (Abcam), anti-p-Akt, anti-Akt, anti-p-S6K1, anti-S6K1, anti-PDCD4 (Cell signaling Technology) and anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE healthcare).

Cells were washed with PBS and lysed on ice for 15 min with lysis buffer (40 mM HEPES, 2 mM EDTA, 0.3 % CHAPS, protease (Sigma) and phosphatase inhibitor mixtures (Thermo Scientific)). Cell lysates were centrifuged at 4 °C for 10 min at 21,000 g, and the supernatants were incubated with anti-FLAG M2 beads (Sigma) overnight at 4 °C. The beads were then washed five times with lysis buffer and the immune-precipitated (IP) protein was collected through boiling of the beads with SDS containing sampling buffer for 10 min. For western blot analysis, cell lysates or the IP proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% milk in TBS buffer containing 0.1 % Tween-20 and subsequently incubated with different antibodies (anti–Dapk2 (Abcam); anti-Flag, anti-HA (Sigma); anti-pPLCγ1 (Y783) (Thermo Scientific); anti-pErk1/2 (T202/Y204), anti-pS6K1 (T389), anti-pAkt (T308), anti-PLCγ1, anti-Erk1/2, anti-S6K1, anti-Akt, anti-Raptor and anti-Actin (Cell Signaling Technology)). The membranes were then stained with matching HRP-conjugated secondary antibodies to allow visualization of target proteins (Jackson Immunoresearch Laboratories). Densitometry for protein bands was analyzed with the AlphaView software (ProteinSimple).

All antibodies were purchased as follows: anti-NEDL2 (ab92711, Abcam), anti-Ret (ab134100, Abcam), anti-Neurofilament (ab50284, Abcam), anti-Akt (sc-8312, Sata Cruze), anti-pAkt (#4060, Cell Signaling), anti-S6K1 (#9202, Cell Signaling), anti-pS6K1 (#9234, Cell Signaling), anti-p85 (#4257, Cell Signaling), anti-p110 (#4249, Cell Signaling), anti-BrdU (#5259, Cell Signaling), anti-Myc (MBL), anti-Flag (MBL), anti-Hsp90 (sc-101494, Santa Cruz).

The whole proteins were extracted with RIPA lysis buffer comprising proteinase and phosphatase inhibitors (Sigma, USA). The whole protein was separated with SDS-PAGE and shifted to a PVDF membrane which was sealed using 5% skimmed milk and probed using antibodies at 4°C all night long. We visualized the protein bands using chemiluminescence analysis and quantified them with ImageJ (NIH, USA). Antibodies comprised in western-blot were anti-AKR1B1 (1:1000, Bioss, China), anti-p-AKT (1:1000, Cell Signaling Technology, USA), anti-p-S6K1 (1:1000, Cell Signaling Technology, USA), and anti-β-actin (1:1000, Bioss, China) antibodies.

Western blots were performed as previously described [35 ]. Briefly, the following antibodies were incubated on membranes overnight: anti-P-S6K1 (1/1000) (Cell Signaling Technology # 9206,) anti-Myogenin (1/100) (Santa-Cruz, sc-576), and anti-REDD1 (1/1000) (ProteinTech 10638-1-AP). The next day, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at 1/3000 for 1 h at room temperature. Membranes were developed with an enhanced chemiluminescent (ECL) reagent from Biorad using a Chemidoc™ Touch Imaging System (BioRad). The stain-free system from Biorad was used to quantify the total protein load and to normalize phosphorylated S6K1.