Chemidoc xrs system image labtm software

The total protein of the individual samples (n = 4) was extracted and quantified by the Bradford assay (Bio-Rad, Hercules, CA, USA) and stored at −70 °C. The protein detection was performed by electrophoresis in SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Blots were blocked with 3% BSA for 60 min at room temperature and incubated overnight at 4 °C with the primary antibodies. The blots were incubated with anti-rabbit, anti-goat, or anti-mouse secondary antibodies conjugated with horseradish peroxidase (1:15,000). GAPDH (1:3500) was used to normalize the data. The images were analyzed with a ChemiDocTM XRS + System Image LabTM Software (Bio-Rad, Hercules, CA, USA). The assays were performed three times using independent blots.

Total protein of pooled kidney samples (n = 4) was extracted and quantified by Bradford assay (Bio-Rad, Hercules, CA, USA) and stored at −70 °C. The protein detection was performed by electrophoresis in SDS-PAGE and then transferred to polyvinylidene difluoride membranes. All blots were blocked with 5% nonfat dry milk (Bio-Rad, Hercules, CA, USA) for 60 min at room temperature and incubated overnight at 4 °C with the following antibodies: toll-like receptor 4 (TLR-4) (1:10,000), nuclear factor-kappa B (NF-κB) (1:3000), and tumor necrosis factor α/β (TNF-α) (1:2000). The blots were incubated with anti-rabbit, anti-goat, or anti-mouse secondary antibodies conjugated with horseradish peroxidase (1:3500). Actin (1:5000) was used to normalize the data. Images were analyzed with a ChemiDocTM XRS + System Image LabTM Software (Bio-Rad, Hercules, CA, USA). The assays were performed three times using independent blots.