Gapdh

Cells were collected and lysed, and the protein concentration was determined. 10% SDS-PAGE gel was added into the protein, and electrophoresis was performed first. After marker separation, 0.45 μm PVDF membrane was transferred between the gel after methanol activated membrane. The protein was separated and transferred to a polyvinylidene difluoride membrane followed by incubation with 5% milk at 20 ± 5 °C for 1 h. The membrane was incubated at 4 °C overnight with the following antibodies: METTL3 (1:1000, AB195352, Abcam, Cambridge, MA, USA), VEGFA (1:1000, AB185238, Abcam, Cambridge, MA, USA), and GAPDH (1:1000, Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China). The secondary antibody was then added. The second antibody was sheep anti-rat and sheep anti-rabbit, and the dilution ratio was 1:5000 and the membrane was incubated at room temperature for 1 h. The protein expression was observed using a chemiluminescence gel imaging system (Tanon 5200, Shanghai, China).

Western blotting was performed according to our previous study [42 (link)]. After treatment with olaquindox, cells were collected and lysed in a lysis buffer containing 20 mMTris–HCl, 4% SDS, 1 mM EDTA, 50 mM NaF, 0.5 mM Na₃VO₄ and 1 mM PMSF at 4 °C for 15 min. Protein in the buffer was loaded into SDS-polyacrylamide gel (SDS-PAGE) for electrophoresis. Then, running gel was transported to nitrocellulose membranes. After being blocked with 5% non-fat milk for 2 h, the membranes were washed with tris buffered saline tween (TBST) and incubated with primary and secondary antibodies. Finally, the membranes were measured by ECL luminescent detection kit (Vigorous Biotechnology). Western blot density was evaluated by the ImageJ 1.46 software (National Institutes of Health, Bethesda, MD, USA). The primary antibodies were performed as followed: rabbit polyclonal antibodies against TSC2, LC3, phosphorylation-p70s6k (p70s6k) (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and Beclin 1 (1:1000; ABclonal Biotech, Cambridge, MA, USA), mouse polyclonal antibodies against GAPDH (1:2000; Zhongshan Golden Bridge, Beijing, China). The secondary antibodies were anti-rabbit IgG (1:5000) and anti-mouse IgG (1:5000) (Zhongshan Golden Bridge).

After determination of the protein concentration, protein samples were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked by 5% non-fat milk in TBST, then incubated at 4 °C overnight with the following primary antibodies: EphB2 (1:1000; R&D Systems, Minneapolis, MN, USA), GluN1 (1:1000; Invitrogen), GluN2A (1:800; Santa Cruz, Dallas, TX, USA), GluN2B (1:1000; Santa Cruz), P-GluN2B (Y1472) (1:1000; Millipore, Bedford, MA, USA), P-EphB2 (Y594) (1:1000; Abcam, Cambridge, MA, USA), β-actin (1:2000; Santa Cruz), GAPDH (1:1000) and His-tag (1:1000; Zhongshan Goldenbridge Biotechnology, Beijing, China). After rinses with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000; Beyotime Institute of Biotechnology, Haimen, China). Protein bands were visualized with an ECL detection system (Beyotime Institute of Biotechnology) and quantified densitometrically with ImageJ software (NIH).