Tumor necrosis factor α tnf α

The RAW264.7 mouse macrophage (Cat#TIB71) cell line was obtained from the American Type Culture Collection. SIM (Cat#PHR1438‑1G) and LPS (Cat#L2630‐10MG) were purchased from Sigma‑Aldrich, Merck KGaA. MCC950 (CP‐456773, Cat# GC31644) was purchased from Glpbio Technology. Fetal bovine serum (FBS, Cat#10099141C) and the α‐modification of Eagle's medium (α‐MEM, Cat#C12571500BT) were purchased from Gibco, Thermo Fisher Scientific, Inc. A tartrate‐resistant acid phosphatase (TRAP) staining kit (Cat#G1492), and 4′,6‐diamidino‐2‐phenylindole (DAPI) and TRITC phalloidin (Cat#CA1610) were purchased from Beijing Solarbio. Bone slice (Cat#Q2356) was purchased from Shanghai Millennium Biology. Specific antibodies against NLRP3 (Cat#23094‐1), ATG7 (Cat#6251), P62 (Cat#4844), Beclin‐1(Cat#19662), caspase‐1 (Cat#16883), tumor necrosis factor‐α (TNF‐α) (Cat#19147), LC3II (Cat#18709) were obtained from Abcam; cleaved‐caspase‐1 (Cat#89332) and IL‐1β (Cat#12507) were obtained from Cell Signaling Technology. Interleukin‐10 (IL‐10) (Cat#60269‐1‐lg). horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit IgG secondary antibody (Cat#L3012) was purchased from Signalway Antibody. Polyvinylidene difluoride (PVDF, ISEQ. 00010) membranes were obtained from EMD Millipore.

Western blotting was performed as described previously [30 (link), 35 (link)], and MT expression was detected using a modified western blotting protocol [24 (link)]. Primary antibodies against the following were used: 3-nitrotyrosine (3-NT; 1 : 1,000; Millipore, Billerica, MA, USA); Nrf2 (1 : 1,000; Abcam, Cambridge, MA, USA); α-smooth muscle actin (α-SMA; 1 : 1,000; Abcam); connective tissue growth factor (CTGF; 1 : 1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); fibronectin (1 : 1,000; Abcam); tumor necrosis factor α (TNF-α; 1 : 1,000; Abcam); NACHT, LRR, and PYD domain-containing protein 3 (NLRP3; 1 : 1,000; Abcam); NAD(P)H:quinone oxidoreductase 1 (NQO1; 1 : 1,000; Santa Cruz Biotechnology); superoxide dismutase-2 (SOD-2; 1 : 5,000; Santa Cruz Biotechnology); catalase (CAT; 1 : 5,000; Santa Cruz Biotechnology); β-actin (1 : 8,000; Santa Cruz Biotechnology); and MT (1 : 1,000; DakoCytomation, Carpinteria, CA, USA).
The protein content was determined by measuring the gray values of bands using Image Lab (Bio-Rad).

Total protein was extracted from kidney tissues using RIPA buffer and separated on 8-10% SDS-PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Then, the membranes were blocked with dried milk (5%) in PBS for 1 h, followed by incubation for 16 h at 4°C with the following primary antibodies: phospho-nuclear factor kappa B (NF-κB) p65 (1:500 dilution) and β-actin (1:1,000 dilution) (Cell Signaling Technology); NF-κB p65 (1:800 dilution) and tumor necrosis factor-α (TNF-α, 1:1,000 dilution) (Abcam); and PAI-1 (1:500 dilution) (Santa Cruz Biotechnology). The following morning, the blots were washed three times with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20 and incubated with the appropriate peroxidase-conjugated secondary antibodies for 1 h. After three washes, the protein bands were identified using ECL (Thermo Scientific, Rockford, IL, USA). Finally, the photographic density was quantified by ImageQuant 5.2 software (Molecular Dynamics).