Ripa buffer

Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (ASPEN Biotechnology, China) for immunoblotting. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), according to the manufacturer's instructions, and bovine serum albumin served as the standard. Samples were resolved via sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and transferred to polyvinylidene difluoride membranes (Millipore, USA). Target bands were visualized using a Tanon-5200 Image Analyser. Protein band intensity was quantified via densitometry using ImageJ 1.48 software (National Institutes of Health, Bethesda, MD, USA). Primary antibodies and secondary antibodies were purchased from Abcam and Cell Signaling Technology. The information and dilution concentrations of the primary antibodies and secondary antibodies are shown in Table 2.Target bands were visualized using a Tanon-5200 Image Analyser. Protein band intensity was quantified via densitometry using ImageJ 1.48 software (National Institutes of Health, Bethesda, MD, USA).

Total proteins in cells were extracted using RIPA buffer (Aspen Biological, Wuhan, China) and protease inhibitors (MedChemExpress, SNJ, USA). Protein concentration was measured using the BCA kit (Aspen). Equal amounts of protein were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA), and blocked with milk at room temperature for 1 h. PVDF membranes were washed three times with TBST for 10 min each, and then incubated overnight at 4 °C with the appropriate primary antibody. Following another round of washing, the membranes were incubated with secondary antibodies at room temperature for 1 h. Finally, the membranes were developed using ChemiDoc XRS gel imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this experiment are listed below: Anti-beta Tubulin (PTM-6414, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys18) (PTM-1427RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys14) (PTM-1414RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl-Histone H3 (Lys9) (PTM-1419RM, PTMBIO, Zhejiang, China); Anti-L-Lactyl Lysine (PTM-1425, PTMBIO, Zhejiang, China); Anti-Histone H3 (PTM-6621, PTMBIO, Zhejiang, China); SLC25A29 (26663-1-AP, Proteintech, Wuhan, China).

NP tissues from three patients in each group were extracted and immediately placed on ice. The tissues were washed with pre-cooled PBS and homogenized using RIPA buffer (Aspen, China) supplemented with phenylmethanesulfonyl fluoride and protease and phosphatase inhibitors (Aspen, China). Tissue lysates were sonicated on ice, and protein concentrations were determined using a BCA protein assay kit (Sigma). Total protein was separated via SDS-PAGE on 10% gels and subsequently transferred onto PVDF membranes (EMD Millipore Corporation, USA) at 250 mA. After blocking with 5% non-fat dried milk in TBST at room temperature for 1 h, the membranes were incubated with anti-Notch1 (Abcam, ab44986), anti-NICD (Abcam, ab83232), anti-Notch2 (CST, 5732), anti-Notch3 (CST, 5276), anti-Notch4 (CST, 2423), anti-HIF-1α (Novus, NB100–105), anti-HES1 (Abcam, ab49170), and anti-GAPDH (ProteinTech, 60,004–1-Ig) primary antibodies overnight at 4 °C. After washing with TBST three times, the protein bands were incubated using HRP-conjugated secondary antibodies (ProteinTech) at room temperature for 1 h. The protein bands were detected using enhanced chemiluminescence detection reagents.