Hpa037364

Paraffin-embedded NPC samples were collected from Xiangya Hospital of Central South University. Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval, and endogenous peroxidases were blocked. The sections were washed three times with 0.01 mol/L PBS (2 mmol/L NaH₂PO₄, 8 mmol/L Na₂HPO₄, and 150 mmol/L NaCl). Then, 0.01 mol/L PBS supplemented with 5% normal goat serum and 0.1% Triton X-100 was used to block the sections, followed by an incubation with anti-YTHDC2 (1:1,000; Sigma, HPA037364) overnight at 4°C. After three times washing in PBS and treatment with adjuvant for 20 min at 37°C, the sections were exposed to secondary antibody for 2 h at 37°C. The immunohistochemical reaction was visualized with 3, 3, 0-diaminobenzidine (DAB) for 3 min. All sections were counterstained with haematoxylin. Both the staining intensity and extent of protein expression were automatically scored by Vectra 2 system (Perkin-Elmer, USA).

NPC cell lines were lysed in a standard RIPA buffer containing proteinase and phosphatase inhibitors (Roche, Indianapolis, IN, USA), and the protein concentrations were quantified using Bradford reagent (Bio-Rad). Then prepared protein was separated via SDS-PAGE and transferred to NC membrane (Pierce Biotechnologies Inc., Rockford, IL, USA). The primary antibodies used in the study include anti-METTL3 (15073-1-AP, Proteintech), METTL14 (HPA038002, Sigma), FTO(ab92821, Abcam), ALKBH5 (HPA007196, Sigma), WTAP (sc-374280, Santa Curz), YTHDF1 (17479-1-AP, Proteintech), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (ab103328, Abcam), YTHDC1 (GTX32976, GeneTex), YTHDC2 (HPA037364, Sigma), IGF1R (3027,CST), AKT (9272, CST), Phospho-AKT (ser473, CST), S6 Ribosomal protein (2217, CST), and Phospho-S6 Ribosomal protein (ser240/244)(2215, CST). The bound antibodies were detected with HRP-conjugated secondary antibodies (Abcam). Densitometric analysis of WB results was performed with ImageJ software.