Anti pten

Cells were washed three times with PBS, lysed with RIPA buffer on ice for half an hour and centrifuged at 12,000 g/s for 20 min to collect the supernatant. We used a BCA kit (Boster Biological Technology) to detect the protein concentration. Then, the proteins (10 µL/lane) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk, probed with primary antibodies (1:1000) and stained with horseradish peroxidase (HRP)-linked secondary antibodies (1:15,000, Promega). Then, the blots were visualized using an ECL kit (Tiangen). The antibodies used in this study were as follows: anti-PTEN (Proteintech, 22034-1-AP), anti-β-actin (Proteintech, 20536-1-AP), and anti-β-Catenin (Proteintech, 66379-1-lg). Anti-p-mTOR (5536), anti-mTOR (2983), anti-P65 (8242), anti-IKB (4814), anti-p-ERK1/2 (4370), anti-ERK1/2 (5013), anti-p-STAT3 (9131), anti-STAT3 (9132), anti-CDK2 (18048), anti-CyclinB1 (12231), anti-P21 (2947), anti-cleaved caspase-3 (9664), anti-cleaved caspase-9 (20750), anti-p-GSK-3β (5558) and anti-GSK-3β (12456) were purchased from Cell Signaling Technology [CST, Danvers, MA (20750)]. Anti-p-JAK2 (ab32101), anti-JAK2 (ab108596), anti-vimentin (ab92547), anti-N-cadherin (ab18203) and anti-E-cadherin (ab40772) were purchased from Abcam (MA, United States).

Cisplatin (cis‐diamminedichloroplatinum II), Etoposide (VP‐16) and MK2206 were purchased from Selleck. Puromycin sulphate was purchased from Beyotime Biotechnology. Anti‐YTHDC1 (CST and Abcam), anti‐m⁶A (Abcam), anti‐PTEN (Proteintech), anti‐phosphorylated AKT (ser473) (CST), anti‐γH2AX (ser139) (CST) and anti‐GAPDH (Beyotime Biotechnology) primary antibodies were used in this study. Goat anti‐mouse and rabbit IgG‐HRP (Abcam) were used as secondary antibodies for Western blots. The secondary antibody for immunohistochemistry (IHC) was obtained from a SP Rabbit & Mouse HRP Kit (Cwbiotech).

After cells and tissues were fully lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Beyotime, Jiangsu, China), protein concentrations were detected using a BCA protein detection kit (Beyotime, Jiangsu, China). Protein samples (30 µg) were separated by electrophoresis on 8%–15% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (#PIVH00010, Merck Millipore, Burlington, MA, USA). After blocking for 2 h, the membranes were incubated with specific primary antibodies (diluted in PBS containing Tween-20) at 4 °C overnight, followed by the respective secondary antibodies at room temperature for 1.5 h. Protein bands were detected using enhanced chemiluminescence detection kit (Merck Millipore) and quantified using Image J software (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA). The used primary antibodies were anti-RBM10 (#14,423‐1‐AP, 1:500), GAPDH (#60,004–1-lg, 1:3000) from Proteintech Group (Chicago, IL, USA); anti-PTEN (#9188 T, 1:1000), anti-PI3K (#4257, 1:500), anti-p-PI3K (#abs130868, 1:500), anti-AKT (#4691, 1:800), anti-p-AKT (#4060, 1:500), anti-mTOR (#2983, 1:1000), anti-p-mTOR (#5536, 1:1000) from Cell Signaling Technology (Danvers, MA, USA).

Cell and tissue proteins were extracted with a RIPA extraction kit (Wanlei, China). A 10% SDS–PAGE gel was used to isolate the same amount of protein (50 μg). The proteins were transferred to a PVDF membrane (Roche, USA). At room temperature, a 2 h sealing was accomplished using nonfat milk (5%). At 4°C, the primary antibodies were incubated overnight, including anti-PTEN (1 : 1000 dilution, Proteintech), anti-PI3K (1 : 1000 dilution, Abmart), anti-p-AKT (1 : 1000 dilution, Abmart), anti-AKT (1 : 2000 dilution, Abmart), anti-α-SMA (1 : 1000 dilution, Wanlei), anti-COL1 (1 : 500 Wanlei), anti-FN (1 : 1000 dilution, Wanlei), and anti-GAPDH (1 : 3000 dilution, Abmart) antibodies. The membranes were nurtured for 1 h using horseradish peroxidase (Wanlei, China) under ambient temperature. The protein bands were detected via an advanced ECL kit (Beyotime, China).