Ripa lysis reagent

Protein levels were performed using western blotting assay. Protein was collected using RIPA lysis reagent (Beyotime Biotechnology, China) with protease inhibition (Thermo Fisher, USA), and the concentrations of protein were measured using a BSA kit (Thermo Fisher, USA). Equal amounts of 30 μg protein were added into each lane, followed separating through 10% gels and transferring to PVDF membranes (Millipore, USA). After blocking with 5% milk, membranes were incubated with primary antibodies (anti-PRDX2, Abcam, ab109367, 1 : 1000 dilution; anti-β-actin, Bioworld, BS6007MH, 1 : 5000 dilution) overnight at 4°C, followed incubating with secondary antibodies (Cell Signaling Technology, 1 : 5000 dilution) for 2 hours at room temperature.

The left kidney was weighed to calculate the kidney index (kidney weight/body weight, mg/g) and then homogenized in RIPA lysis reagent (Beyotime, Shanghai, China). The levels of TNF-α, IL-6, COI IV, and transforming growth factor (TGF)-β1 in the renal homogenates were measured using ELISA kits (Boatman, Shanghai, China).

Tissue and cell specimens were lysed by RIPA lysis reagent (Beyotime, China) on the ice for half hour and sonicated in an ice bath for 3 min. Following centrifugation at 4°C at 12,000 r/min for 10 min, the supernatant was harvested. The protein concentrations were calculated with BCA kit (Beyotime, China). Then, lysate was boiled with 5 × SDS loading buffer at 100°C for 5 min. Then, protein was separated by SDS-PAGE electrophoresis as well as transferred onto PVDF membranes (Millipore, Germany). Following being washed with TBST, the membranes were blocked by 5% milk/TBST lasting 1 h. Then, the membranes were probed with primary antibodies against METTL3 (1 : 1000; 15073-1-AP; Proteintech, Wuhan, China), ZC3H13 (1 : 1000; DF4623; AFFINITY, USA), YTHDF2 (1 : 1000; 24744-1-AP; Proteintech, Wuhan, China), or GAPDH (1 : 5000; ATA00013Rb; AtaGenix, Wuhan, China) at 4°C overnight. The membranes were washed by PBST for three times. Afterwards, the membranes were exposed to HRP-labeled goat antirabbit secondary antibody (SA00001-2; Proteintech, Wuhan, China) at room temperature for 1 h. The membranes were developed with luminescent buffer and investigated by ChemiDoc™ XRS + gel imaging system (Bio-Rad, Shanghai, China).

Radioimmunoprecipitation assay (RIPA) lysis reagent (Beyotime) was used for protein extraction, followed by protein quantification using the BCA kit (Beyotime). Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE; 10%) was performed to separate the proteins before transferring the protein bands onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked using a blocking buffer (Beyotime) for 30 min. They were then incubated overnight at 4°C with primary antibodies targeting MGMT (ab108630; Abcam, Cambridge, MA, USA; 1/2000 dilution) or GAPDH (ab9485; 1/2500 dilution). The membranes were then incubated with a secondary antibody (ab205718; Abcam; 1/5000 dilution) for 1.5 h at room temperature. Finally, the protein signals were visualized using the enhanced chemiluminescence (ECL) kit (Beyotime) [31 (link)].

Total proteins were extracted using RIPA lysis reagent (Beyotime, Shanghai, China) and the protein contents were measured via BCA kit (Beyotime, Shanghai, China) as per the instructions. Protein samples were separated through SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). After blocking, membranes were labeled with primary antibodies (anti-PTEN, 1:1000, ab245322; N-cadherin, 1:1500, ab19348; E-cadherin, 1:2000, ab133597; GAPDH, 1:2500, ab245355; Vimentin, 1:1000, ab92547; all purchased from Abcam, Cambridge, USA) overnight at 4 °C. Following TBST washing, the membranes were subjected to 2-h incubation with HRP-labeled secondary antibody (1:3000, ab205718) at ambient temperature. Protein band visualization was achieved with an ECL detection kit (Yeasen, Shanghai, China).