Anti gapdh

Cells on 6-well plates were rinsed twice with cold PBS and lysed in RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxycholicphenyl methylsulfonyl fluoride, 5.0 mM sodium pyrophosphate, 1.0 g/mL leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and1 mM DTT) containing a protease inhibitor (PMSF) mixture at 1:100 dilution on ice for 30 min. The insoluble components of cell lysates were removed by centrifugation (4 ^oC, 12000 × g, 10 min), and protein concentrations were measured using a Pierce BCA protein assay kit. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked using 5% skim milk in TBST and then incubated with diluted indicated primary antibodies: anti-GPX4 (1:1000 dilution, Proteintech, Cat No: 30388-1-AP), anti-SLC7A11 (1:1000 dilution, ABclonal, Cat No: A13685), anti-GAPDH (1:1000 dilution, Wanlei, Cat No: WL01114) at 4 ^oC with gentle shaking overnight. After washing five times (5 min each) with TBST, membranes were incubated with the HRP conjugated goat anti rabbit IgG (H + L) (1:5000 dilution, Wanlei, Cat No: WLA023) for 1 h at rt. Immunoreactive bands were visualized using an ECL detection kit (Wanlei, Cat No: WLA006) following the manufacturer’s instruction on the ChemiDoc system (BioRad, Shanghai, China).

Protein was extracted from renal tissue using RIPA reagent, and the total protein content was determined by a BCA protein quantitative kit (Solarbio Biotechnology, Beijing, China). SDS–PAGE (TransGen Biotech, Beijing, China) was followed by film transfer and closure and incubation with primary and secondary antibodies; rabbit pAb anti-GAPDH (1:1000, Wanleibio, Shenyang, China), rabbit pAb anti-P65 (1:500, Wanleibio, Shenyang, China), rabbit pAb anti-IFN-γ (1:1000, Wanleibio, Shenyang, China), rabbit pAb anti-IL-6 (1:1000, Wanleibio, Shenyang, China), and rabbit pAb anti-IL-8 (1:1000, Wanleibio), followed by the corresponding HRP-conjugated secondary antibodies (1:5000, Bioss, Beijing, China). Then, the signal was detected with a Bio–Rad Chemidoc Touch imager (Bio–Rad Chemidoc Touch, CA, USA). Finally, the gray value of the corresponding protein was analyzed by ImageJ software (National Institute of Health, Bethesda, Maryland, USA).

Cell lysates were harvested. Total protein of equivalent amounts were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE). After that, they were transferred to polyvinylidene difluoride (PVDF) membranes. Block the membranes with 5% fat-free milk and 0.1% Tween-20 in tris-buffered saline with Tween (TBST) for 1.5 h. Next, the membranes were incubated with diluted anti-Notch1 (Abcam), E-cadherin (CST), N-cadherin (CST), Vimentin (Abcam), Fibronectin (Abcam), Snail-1 (Wanleibio), Shh (CST) and anti-GAPDH (Wanleibio) primary antibodies. Anti-rabbit or anti-mouse secondary antibodies (ZSGB-BIO), which were horseradish peroxidase-conjugated, were used and detected by the ECL system (Fujifilm Las-4000).