Quantity one

Cells after 7 days of osteogenic induction were lysed in RIPA buffer (Beyotime, China) on ice. The samples were heated at 95 °C for 5 min in a sample buffer containing 20% SDS-PAGE sample loading buffer, separated on 10% SDS-polyacrylamide gels, and transferred to the PVDF membranes by a wet transfer apparatus (Bio-Rad). The membranes were blocked with 5% BSA for 1 h and then incubated overnight with rabbit METTL3 polyclonal antibody (1:1000, Cell Signaling Technology, Cat No: 96391), METTL14 polyclonal antibody (1:1000, ABclonal, Cat No: A8530), FTO polyclonal antibody (1:1000, Affinity, Cat No: DF8421), ALKBH5 polyclonal antibody (1:2000, Proteintech, Cat No: 16837-1-AP), and α-tubulin rabbit polyclonal antibody (1:2000, Beyotime, Cat No: AF0001), followed by incubation with a goat anti-rabbit IgG secondary antibody HRP conjugated (1:5000, Signaling antibody, Cat No: L3012-2). The antibody-antigen complexes were visualized with Bio-Rad Quantity One (Bio-Rad Laboratories Inc., Hercules, CA, USA).

A total of 25 ml of each cold-shocked R15 culture, which was cultured for different durations, was harvested at 12,000 g at 4°C for 3 min, resuspended in 400 μL PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4), and lysed by ultrasonication at 240 W in a cycle of 3 s sonication and 3 s pause for 15 min on ice. All manipulations were conducted following standard protocols (Sambrook and Russell, 2001 ) with a few modifications. Total cellular protein was electrophoresed on Tricine-SDS-PAGE below 100 mA for 1.5 h and transferred to a 0.1-μm nitrocellulose filter membrane (Easybio, Beijing, China) under 100 V for 1 h. To fix the proteins to the membrane surface, ddH₂O and 1 × TBS (2.42 g Tris, 8.0 g NaCl in 1 L, pH 7.6) were used to wash the membrane with gentle shaking for 5 min, and 0.2% glutaraldehyde diluted in 1 × TBS was used to wash the membrane for 45 min. The TRAM3066-specific peptide and TRM2002 total protein-generated antibodies were prepared by the MBL (Beijing Biotech Co., Ltd.). The secondary antibody was purchased from Easybio (Beijing, China).
The density of the Western blotting protein band was determined using Bio-Rad Quantity One (Bio-Rad, Hercules, CA, United States), and the amount of protein was calculated by referring to the gray value of the overexpressed protein.

The protocol for determining the protein abundance of NOS and RAS components via Western blot analysis was based as per our previous reports [19 (link)]. Equal amounts (200 μg) of kidney cortical protein extracts were loaded. After the separation of proteins via SDS-PAGE, the proteins were then transferred onto nitrocellulose membranes. For this, we used Ponceau S red (PonS) as the loading controls. Following staining with the PonS stain solution (0.2% w/v in 1% acetic acid), the membranes were imaged and saved in the TIFF file for the purposes of quantitative analysis. After washing the membranes, the nonspecific sites were blocked through incubation in the blocking buffer (5% milk in 0.05% TBS-T). Later, the membranes were incubated with the indicated primary antibodies, as listed in Table 1. Following the incubation loaded with a secondary antibody, the immuno-reactive bands were pictured using enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA) and digitized by Bio-Rad Quantity One (Bio-Rad, Hercules, CA, USA). In this regard, the protein abundance was given as the integrated optical density (IOD)/PonS.