Electrochemiluminescence reagent

In brief, add 1 ml of lysate plus 10 μl of PMSF (100 mM) to cells and tissue, after lysing for 30 min; the lysate can be transferred to a 1.5 ml centrifuge tube with a pipette, then centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant is placed in a 0.5 ml centrifuge tube and placed at -20°C. 30 to 60 μg of protein/lane was subjected to electrophoresis by using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). The resolved proteins were transferred electrophoretically to nitrocellulose membrane “blots.” The blots were blocked with 5% BSA in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 5% BSA in TBST with anti-Klotho (1 : 1,000; Cell Signaling), anti-TLR4 (1 : 1,000; Abcam), and anti-NF-KB (1 : 1,000; Cell Signaling). Immunolabeling was detected with electrochemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA). Quantification of Western blots was performed by using ImageJ pixel analysis (NIH Image software). Data from Western blots are presented as band density normalized to the loading control (actin).

A nuclear extract kit (Active Motif, Tokyo, Japan) was used to extract cytoplasmic and nuclear proteins of kidney tissues, according to the manufacturer's protocol. Protein extracts separated upon 7.5% SDS-PAGE were transferred to 0.45 m PVDF membrane (Bio-Rad, California, Hercules, USA). The membranes were incubated with TBST (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) with 5% nonfat milk powder for 2 hours before Western blotting overnight at 4°C with rabbit polyclonal antibodies against mouse Nrf2, HO-1, NQO-1, GSR (Cell Signaling Technology, USA), and GCLc (av54576, Sigma, USA). Histone H3 and β-actin (Cell Signaling Technology, USA) were used as a protein control to normalize the volume of protein expression. After being washed for 3 × 10 minutes with TBST, the membranes were incubated with HRP-conjugated secondary antibody (1 : 2000, Cell Signaling Technology, USA) for 1.5 hours. Then, after the membranes were washed 3 times with TBST, immunoreactive bands were visualized with electrochemiluminescence reagent (Amersham, Uppsala, Sweden). Densitometric and ImageQuant analyses were subsequently quantified using Image Lab Software (Bio-Rad, USA).

Total protein concentration was calculated by the BCA Protein Assay kit (Pierce, Rockford, IL, USA), according to the manufacturer's instructions. The cells and tissues were lysed in cell lysis buffer for 20 min vibration on the ice and centrifuged at 12.000 × g at 4°C for 15 min. Proteins were separated upon 10% SDS-PAGE and transferred onto 0.45 mm PVDF membrane (Bio-Rad, California, Hercules, USA). The membranes were incubated with 5% nonfat milk powder which dissolved in TBST (20 mM Tris-HCL, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 2 hours. The following protein antibody Nrf2 (68.0 kDa), Histone H3 (15.0 kDa), and GAPDH (40.2 kDa) (Cell Signaling Technology, CST); HO-1 (34.6 kDa), NQO-1 (30.0 kDa), and GST (24.0 kDa) (Abcam); and ANXA5 (36.0 kDa) (Santa Cruz, CA) were used to bind with corresponding proteins and incubated overnight at 4°C. All these protein antibodies were diluted by the Antibody Dilution Buffer (Beyotime, China) with a ratio of 1 : 1000-2000. HRP-conjugated secondary antibody (1 : 4000, Cell Signaling Technology, USA) was used to bind with primary antibodies for about 1.5 hours. Finally, immunoreactive bands were visualized with electrochemiluminescence reagent (Amersham, Uppsala, Sweden). Densitometry and quantitative analysis were analyzed using the ImageJ software (Bethesda, USA) and regulated by Histone H3 and GAPDH.