Ab232731

0.1 g of renal tissue was added with 1 mL of RIPA lysate (Beyotime Institute of Biotechnology) and placed on ice, and homogenized thoroughly by a homogenizer. The protein concentration was then detected using a BCA kit (Bio-Rad Laboratories, Inc.). The denatured proteins (30 μg) were separated by SDS-PAGE gel electrophoresis. The isolated proteins were transferred to PVDF membrane and sealed with 5% skimmed milk powder for 2 h. The proteins were incubated overnight in primary antibody at 4 °C. On the second day, PVDF membrane was incubated in the secondary antibody for 2 h and developed with enhanced chemiluminescence kit(GE Healthcare) and Image J software (version 146; National Institutes of Health, Bethesda, MD, USA) was used to analyze the fold-changes of protein levels. The primary antibody information is as follows:anti- KIM1 (ab66062, Abcam, UK), anti-NGAL (ab23477, Abcam, UK), anti- SLC7A11 (ab175186, Abcam, UK), anti- GPX4 (ab125006, Abcam, UK), anti- FTH1 (ab75972, Abcam, UK), anti- FPN1 (ab78066, Abcam, UK), anti- Nrf2 (ab62352, Abcam, UK), anti- HO-1 (ab68477, Abcam, UK), anti- Lamin B (ab232731, Abcam, UK), anti-GAPDH (ab8245, Abcam, UK).

After infection for 12 h, U937 cells were collected and lysed with lysis buffer, and the protein concentration was measured with a Bradford protein assay kit. Thirty micrograms of total protein were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked by 5% BSA for 1 h at room temperature, and incubated with primary antibodies, including Rabbit monoclonal anti-SQSTM1/p62 (1:1000, ab109012, Abcam), Rabbit monoclonal anti-Caspase-3 antibody (1:1000, ab32351, Abcam), Rabbit monoclonal anti-Cleaved Caspase-3 antibody (1:1000, ab32042, Abcam), Mouse monoclonal anti- Lamin B antibody (1:1000, ab232731, Abcam), Mouse monoclonal antibody anti-β-actin (1:1000, ab8226, Abcam) and human polyclonal antibody anti-GAPDH (1:1000, ab8245, Abcam) at 4 °C overnight. Then the membranes were incubated with specific secondary antibody goat Anti-mouse IgG (1:1000, ab150113, Abcam) for 2 h at room temperature. All immunoblots were visualized with chemiluminescence reagents (Millipore, Billerica, MA, USA). The protein levels were quantitated by a ChemiDoc XRS Imaging System (Bio-Rad Laboratories, Hercules, CA).

Recombinant mouse TNF-α was acquired from Sigma-Aldrich (654245, St. Louis, MO, USA), while antibodies against Angptl4 were purchased from Sigma-Aldrich (AB10605, St Louis, MO, USA). Antibodies against P65(80979-1-RR), MMP3(18165-1-AP), BAX (50599-2-Ig), Bcl-2(26593-1-AP), cleaved caspase3(19677-1-AP), and Cytochrome c(10993-1-AP) were obtained from Proteintech (Rosemont, IL, USA), while antibodies against C-caspase 3(9661S), P-P65(3039S), and IκBα(9242S) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against GAPDH (ab8245), IL-6 (ab290735), ADAMTS5 (ab41037), Collagen II (ab34712), P-IκBα(ab133462), MMP13 (ab39012), SIRTUIN 1 (ab110304), and Lamin B (ab232731) were obtained from Abcam (Cambridge, MA, USA).