Hrp labeled goat anti mouse igg

Proteins in kidney tissues and HK2 cells were extracted using RIPA lysis buffer (#P0013B, Beyotime, Shanghai, China) containing 1% PMSF (P0100, Solarbio, Beijing, China). The proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes (R7CA6580A, Thermo Fisher Scientific, Waltham, MA). The membranes were probed overnight with GPX4 (1:1000, ab125066, Abcam, Cambridge, MA), and NCOA4 (1:1000, abs-134557, Absin, Shanghai, China). The membranes were respectively incubated with HRP-labeled goat anti-rabbit IgG (1:5000, 511203, Zen BioScience, Research Triangle Park, NC) and HRP labeled goat anti-mouse IgG (1:5000, 511103, Zen BioScience). Bands were detected using Immobilon Western Chemilum HRP Substrate (Catalogue number WBKLS0500, EMD Millipore, Burlington, MA), and protein levels were normalized against β-actin (1:5000; HRP-60008, Proteintech, Rosemont, IL).

The total protein of WT and SNAC9 mutant tomato leaves was extracted simultaneously, and the protein samples were denatured after the total protein content was determined. According to the molecular weight of the SNAC9 protein, the corresponding separation gel and concentrated gel were prepared using an SDS-PAGE rapid gel preparation kit, separated by electrophoresis, and transferred to a PVDF membrane. Mice were immunized with purified recombinant His-SNAC9 protein to obtain anti-SNAC9 polyclonal antibodies (Dynamiker Biotechnology Co., Ltd, Tianjin, China) and incubated with the target protein. Anti-β-actin mouse mAb (Zen-Bioscience, China) was used as the internal reference. The corresponding anti-mouse antibody (HRP-labeled goat-anti-mouse IgG, Zen-Bioscience, China) was selected as the secondary antibody. Following ECL staining, the pictures were examined under an optical microscope, and ImageJ software was used to assess the gray values.