Horse radish peroxidase goat anti rabbit igg

Cells were lysed using a radio immunoprecipitation assay (RIPA) buffer (Boster, Wuhan, China) containing a protease inhibitor. The total protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA) following the procedure outlined in the instructions. Proteins were separated using 12.5% polyacrylamide gel and transferred to 0.45 μm of polyvinylidene fluoride (PVDF) membranes using a Trans-blot Turbo device (Bio-Rad, Hercules, CA, USA) at 75 V on ice for 1.5 h. The membranes were blocked with 5% skimmed milk (Solarbio, Beijing, China) for 1 h at room temperature. Then, the primary antibody was used to incubate the membranes overnight at 4 °C. The primary antibody and dilution rate are described as follows: anti-NRAMP1 (1:500) (Novus, Wuhan, China), β-actin (1:20,000) (Proteintech, 66009-1-Ig, Rosemont, IL), caspase-3 (1:1000) (Abclonal, Wuhan, China), proliferating cell nuclear antigene (PCNA) (1:5000) (Elabscience Biotechnology Co. Ltd., Wuhan, China). Next, the membranes were incubated with the secondary antibody horse radish peroxidase (HRP) goat anti-rabbit IgG (1:10,000) and HRP goat anti-mouse IgG (1:10,000) (ABclonal, Wuhan, China) at room temperature for 1 h. Finally, the membranes were scanned using chemiluminescence imaging software (AI600 Control. RDP).

The plasma protein concentrations were determined by using a nondenaturing gel. Briefly, the plasma was diluted with 1X PBS, and nondenaturing loading buffer (50% glycerol, 0.3 mol/L pH = 6.8 Tris-HCl, 10% SDS, 0.01% bromophenol blue) was added to the samples. To detect the monomeric form of cystatin F, loading buffer with 20% dithiothreitol (DTT) (Sangon biotech, shanghai, China, Cat No. A620058) was used, and the samples were boiled at 95 °C for 10 min. Coomassie Brilliant Blue (CBB) staining of the plasma proteins served as an internal control. Cells were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Biotech, Beijing, China, Cat No. P0013B) containing 1 mM PMSF (Beyotime Biotech, Beijing, China, Cat No.ST506). Then, the protein samples were separated on 12% SDS polyacrylamide gels. For western blot detection, the primary antibody against cystatin F (ABclonal Technology, Wuhan, China, Cat No. A8164) was diluted 1:1000, and Horseradish Peroxidase (HRP) goat anti-rabbit IgG (ABclonal Technology, Wuhan, China, Cat No. AS014) was diluted 1:10,000. Immunoreactive bands were visualized with the SuperSignal West Pico chemiluminescent substrate (Pierce, IL, USA) using an LAS-3000 mini (Fujifilm, Tokyo, Japan). For quantitative analysis, the mean density of each band was measured by ImageJ software.