Anti p 1 κbα

Anti-NOX-2, anti-Rac-1, anti-p47phox, anti-p-53, anti-Bax, anti-Bcl-2, anti-cytochrome c, anti-β-actin, anti-p-I-κBα, anti-p-p38, anti-p-NF-κB, anti-COX-2, anti-IL-8, anti-TGFβ1, anti-p-ERK, anti-Sp1, anti-CTGF, anti-FGF2, anti-uPA, anti-MMP-2, anti-MMP-9, and anti-α-SMA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA).

Immunostaining of γH2AX, p53, and NF-kB was followed by instructed protocol. In brief, cultured glomerular endothelial cells were fixed in 4% PFA for 15 minutes, permeabilized with 0.5% Triton X-100 in PBS for 15 minutes, and blocked with 3% bovine serum albumin in PBS for 60 minutes. All the procedures were performed at room temperature. Cells were then incubated with primary antibody, anti-γH2AX (1:500, rabbit monoclonal, 9718, Cell Signaling Technology, MA, USA), anti-p53 (1:50, rabbit polyclonal, 10442-1-AP, Proteintech, IL, USA), anti-NF-kB (1:100, rabbit polyclonal, PA5-16545, Thermo Fisher, IL, USA), and anti-p-IκBα (1:50, mouse monoclonal, santa cruz, sc-8404, TX, USA) at 4 °C overnight. Cells were incubated with Alexa Fluor Plus 488 goat anti-rabbit IgG secondary antibody (1:500, A32731, Thermo Fisher Scientific, Tokyo, Japan), at room temperature for 1 hour. Nuclei were counterstained with DAPI (H-1200, Vector Laboratories, CA, USA).