Anti keap1

Western blot analysis was performed in cytosolic and nuclear extracts prepared from liver homogenates. The supernatant fraction was collected and stored at -80°C in aliquots until use. Protein concentration was measured by the Bradford assay [20 (link)]. Lysate proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TTBS) for 1 h at room temperature and probed overnight at 4°C with polyclonal anti-NQO1, anti-Keap1, anti-Nrf2, anti-ATF6, and anti BIP/GRP78 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1 : 200-1 : 1.000 dilution with TTBS in 5% nonfat dry milk. Bound primary antibody was detected (HRP—with anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG antibodies) (Sigma-Aldrich, St. Louis, MO, USA). Protein detection was performed via chemiluminescence using a commercial ECL kit (Amersham Pharmacia Biotech, Little Chalfont, Great Britain) [23 (link)]. The density of the specific bands was quantified with imaging densitometer software (Scion Image, Maryland, MA).

Western blotting was performed in accordance with standard procedures, as described previously [2 (link)]. Anti-occludin, anti-zonula occludens-1 (ZO-1), anti-Nrf2, anti-NQO1, and anti-Keap1 (Santa Cruz Biotechnology, Inc.) antibodies and secondary antibody (1 : 2000; Millipore) were used to detect targeted protein expression. Anti-β-actin antibody (Proteintech) was used at 1 : 5000. Images were acquired by a Tanon 5500 imaging system (Tanon, Shanghai). ImageJ software was used to quantitate the staining intensity in the images, and the data were normalized to the sham or control values.

The renal tissues were homogenized in RIPA buffer with a tissue homogenizer. The homogenate was centrifuged at 12,000 rpm for 15 min. HK2 cells were lysed in RIPA Lysis Buffer. Equal amounts of extracted proteins were separated on 10% SDS-PAGE and transferred to a PVDF membrane, blocked with blocking solution for 2 h at room temperature, and probed with the appropriate primary antibodies including anti-Nrf2 antibody (catalog number: #12721, Cell Signaling Technology, Danvers, MA, USA), anti-keap1(catalog number: sc-365626), anti SOD1(catalog number: sc-101523), anti-GAPDH (catalog number: sc- 47724), HO-1 (catalog number: sc-136256) antibodies (Santa Cruz, Dallas, TX, USA), anti-SOD2 (catalog number: 24127-1-AP), and β-actin (catalog number:66009-1) antibodies (Proteintech, Chicago, IL, USA) at 4 °C overnight. This was followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h. Immunoreactive bands were visualized via the enhanced chemiluminescence and quantified by scanning densitometry with Image J gel analysis software. All experiments were performed in triplicate.

To detect the ubiquitination levels of endogenous Nrf2 or Keap1, cells were transfected with or without indicated plasmids for different experimental purpose and treated with 10 μM MG132 (Sigma) for 4 h to block proteasomal degradation. The cells were lysed and immunoprecipitated for 4 h at 4 °C with Protein G Magnetic beads (Thermo) loaded or bound with anti-Keap1 (Santa Cruz), anti-Nrf2 (Abcam), or anti-Myc (Santa Cruz) antibodies according to immunoprecipitation assay described above. The immunoprecipitated proteins were subjected to immunoblotting analysis with antibody against Ubiquitin (Santa Cruz).