Anti fn antibody

We used the BCA Protein Detection Kit (Pierce Biotechnology, USA) to determine the concentration of each protein sample. Western blotting was then carried out in accordance with standard procedures. Membranes were incubated overnight withspecific primary antibodies, as follows: anti-Fn antibody (Abcam, 1:1500), anti-CoL-1 antibody (Affinity, 1:1000), anti-Sp1 antibody (Abcam, 1:5000), and anti-β-actin antibody (Proteintech, 1:3000). The next morning, the membranes were washed and incubated for 1 h with peroxidase-conjugated goat anti-rabbit immunoglobulin G (secondary antibody). Membrane binding to the antibodies was detected with the enhanced chemiluminescence system. Finally, band intensity was evaluated by Image J software (National Institutes of Health, Bethesda, MD).

The Western Blot assay was performed as previously reported [24 (link)]. Briefly, total proteins were extracted from the rat kidney tissues and HK-2 cells from each group. Samples were separated by 10% SDS-PAGE and then transferred into a nitrocellulose membrane (Whatman, Florham Park, NJ). The membrane was blocked using 5% dry fat-free milk in PBS with 0.1% Tween for 60 min. at room temperature, and then incubated with primary antibodies overnight which are shown as follows: anti-α-SMA antibody (1:1000; Abcam, USA); anti-E-cadherin antibody (1:1000; Abcam, USA); anti-FN antibody (1:1000; Abcam, USA); anti-CD147 antibody (1:1000; Abcam, USA); anti-CyPA antibody (1:1000; Abcam, USA); anti-GAPDH antibody (1:1000; Abcam, USA); anti-phospho-p38 MAPK antibody (1:1000; Cell Signaling Technology, USA); and anti-p38 MAPK antibody (1:1000; Cell Signaling Technology, USA). The secondary antibody used was a goat anti-rabbit IgG (1:4000; Abcam, USA). The proteins were visualized by chemiluminescence reagents (Amersham Biosciences, USA). All the experiments were carried out at least three times.