Ecl chemiluminescent substrate kit

Cells and renal tissue were lysed in cold RIPA buffer containing protease inhibitor cocktails. The total protein concentrations were quantified using a BCA protein assay kit. Equal amounts of total protein were separated in 10–15% SDS-PAGE, and samples were transferred onto nitrocellulose membranes, blocked with 5% skimmed milk in TBS/0.5% Tween (TBST) for 1 hour, washed with TBST for three times, and incubated with primary antibodies against MFN2 (1 : 500, Affinity Biosciences), UCP2 (1 : 500, Affinity Biosciences), NRF2 (1 : 500, Proteintech), Slc7a11 (1 : 1000, Abcam), GPX4 (1 : 500, Proteintech), and β-actin (1 : 1000, Proteintech) overnight at 4°C. The nitrocellulose membranes were washed with TBST for three times, 10 min each, and then incubated with secondary antibodies (1 : 10000, Proteintech) for 1 h at room temperature and then washed with TBST for three times again and added with the enhanced ECL Chemiluminescent Substrate Kit (Yeasen Biotechnology, Shanghai, China) in the membrane. The relative intensity of the target band was detected using the AmerSham Imager 600 image system (GE, USA). Images were quantified using ImageJ software (NIH, USA).

Cell lysis buffer for Western and IP (P0013, Beyotime) was used to lyse HGC27 and AGS cells after they had been exposed to various doses of NTP for 24 h. The BCA protein Colorimetric Assay Kit (E-BC-K318-M, Elabscience) was subsequently utilized to determine the total protein. After the protein was separated by electrophoresis, the membrane was transferred using polyvinylidene difluoride (PVDF) at an ice bath temperature. Five percent skim milk was used to block the portions. In a 4 °C shaker, the matching antibodies were incubated overnight. After rinsing with Tris-buffered saline and Tween (TBST), the film was treated with secondary antibodies (S0002, Affinity) for 1.5 h at room temperature. Finally, the imaging system was used to build the improved ECL chemiluminescent substrate kit (36222, Yeasen). For some of the PVDF membranes, Western blot fast stripping buffer (PS107, Epizyme) was used. After completing Western blot luminescence detection, the PVDF membranes were rinsed with TBST for 5 min. Add 10 mL of fast stripping buffer to cover the membrane and rinse for 20 min. The stripping solution was removed, and TBST was added to rinse the PVDF membrane for 5 min. The membrane was sealed with skim milk powder for 2 h, the PVDF membrane was washed with TBST, and then the other antibodies were added again. The gray value was analyzed and calculated using ImageJ software.