β tublin

Bronchoalveolar lavage fluid (BALF) was obtained by instilling 1 ml PBS through the tracheal cannula and suctioning back with a volume of 0.8–0.9 ml. The BALF was centrifuged at 12,000 g for 20 min at 4 °C as soon as finish the collection, and the supernatant was stored at −80 °C. The protein concentration was measured by the bicinchoninic acid (BCA) method using enhanced BCA protein assay kit (Beyotime, Shang Hai, China). The samples were separated using 10% SDS-PAGE and electro-transferred onto nitrocellulose membrane (Bio-Rad, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and were then incubated with primary antibodies in 5% BSA (in TBS + 0.1%Tween 20) overnight at 4 °C. The β-tublin (1:3000,Santa cruz, USA) was used as a loading control. The membranes were washed three times for 5 min each in TBS + 0.1%Tween 20, and were then incubated in the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa cruz, USA) for 2 h at room temperature. Finally, the protein bands were visualized using enhanced chemiluminescence (ECL). The relative quantity of proteins was analyzed using Image J and normalized to that of loading controls. Antibodies included: rabbit anti-TNF-α (1:1000, Abcam, USA), mouse anti-AQP4 (1:1000, Abcam, USA), rabbit anti -β-tublin (1:1000, Santa cruz, USA).

NRK-49F cells were pretreated with Mefunidone (0.18mM) or PFD (2mM) for 1h and then stimulated with 10ng/ml of recombinant human TGF-β1 for 48h. HK-2 cells were pretreated with Mefunidone at different concentrations (0.06, 0.12, and 0.18mM) for 24h or inhibitors for 1–2h and then stimulated with 10ng/ml of recombinant human TNF-α for 15, 30, or 60min. Primary peritoneal macrophages were pretreated with Mefunidone (0.18mM) and different inhibitors for 1–2h and then stimulated with 250ng/ml of LPS for 15, 30, or 60min.
Whole proteins from kidneys or cells were collected as described previously [33 (link)]. Membranes were incubated overnight at 4°C with the following specific primary antibodies against α-SMA (1:2,500; Sigma), fibronectin (1:400; Santa Cruz Biotechnology, Dallas, TX), phosphor-ERK1/2 (1:1,000; Cell Signaling Technology, Danvers, MA), ERK1/2 (1:1,500; CST), phosphor-IκB (1:1,000; CST), IκB (1:1,000; CST), phosphor-STAT3 (Ser727, 1:1000; CST), STAT3 (1:1000; CST) or β-tublin (1:1,000; Santa Cruz).

Cell lysates were prepared by using a RIPA buffer containing Sigmafast Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 3 (Sigma). Protein concentrations were quantified by using the bicinchoninic acid assay (Thermo Scientific). Proteins were separated by SDS PAGE with 10% acrylamide, transferred onto PVDF membranes and incubated with antibodies against SBP1 (1: 1000, Abcam), GPX1 and GPX4 (1:1000, GeneTex), and β-tublin (1: 10,000, Santa Cruz) overnight at 4°C. After being washed in a TBST buffer, the membranes were blocked in 5% non-fat milk (Bio-Rad) for 1 h at room temperature. After secondary antibody incubation, protein levels were represented as chemiluminescence signals after incubation with Supersignal Femto solutions (Pierce) and detected by using a ChemiDoc™ MP Imaging System (Bio-Rad). Band intensity was semi-quantified by using WCIF ImageJ 1.37c. The relative values were normalized with that of β-tubulin.