Radioimmune precipitation assay buffer

For whole cell extraction, cells and tissue samples were lysed in radioimmune precipitation assay buffer (Beyotime, Beijing, China) supplemented with protease and phosphatase inhibitor. Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After being blocked with 5% nonfat dry milk, membranes were incubated overnight at 4°C with primary antibodies against VCAM-1, E-selectin, ERK, P-ERK, JNK1/2, p-JNK1/2, TNF-R1, TRAF2, NF-κB/P65, RIP, GAPDH, and beta-actin that were diluted at the indicated concentration. Then, the membranes were washed three times with TBST (10 mmol/L Tris-HCl, pH 8.0, containing 150 mmol/L NaCl and 0.1% Tween-20) and incubated with secondary antibodies (Merck Millipore, Darmstadt, Germany) for 1.5 h at room temperature. After washing for 3 × 10 min with TBST, target bands were developed using ECL (Advansta, California, USA) and exposed on a film (Kodak, NY, USA). The intensity of the protein bands was measured using Quantity One version 4.6.2 Image software (Bio-Rad, USA).
The anti-GAPDH primary antibody was purchased from Bioworld (St. Louis Park, MN, USA). The anti-p-JNK1/2, TRAF2, NF-κB/P65, and beta-actin primary antibodies were purchased from Abcam (MA, USA). All other primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Radio immune precipitation assay buffer (Beyotime, Shanghai, China) supplemented with 10% phenylmethane sulfonyl fluoride protease inhibitor (Beyotime, Shanghai, China) was used to extract protein in tissues and cells under the guidance of the manufacturer’s direction. Proteins after denaturation were loaded into the sample holes of 10% SDS-PAGE and separated under a stable voltage. Nitrocellulose membranes (Whatman, Maidstone, UK) were put on the surface of 10% SDS-PAGE and transferred protein under a constant electricity. Then, antibodies were utilized to combine the antigens.
The antibodies and their dilutions used for Western blots were as follows: Rabbit Anti-ALDH1A1 antibody (GTX123973; GeneTex, San Antonio, TX, USA; 1:5000), Rabbit Anti-CEBP Beta Polyclonal Antibody (bs-1396R; Bioss, Beijing, China; 1:500) [39 ,40 (link)], Rabbit Anti-PPAR Gamma Polyclonal Antibody (bs-0530R; Bioss, China; 1:500), Rabbit Anti-Adiponectin Polyclonal Antibody (bs-0471R; Bioss, Beijing, China; 1:500) [41 (link)], Rabbit Anti-GAPDH (AB-P-R 001; Hangzhou Goodhere Biotechnology Co. Ltd., Hangzhou, China; 1:10,000), Goat Anti-rabbit IgG-HRP(BA1054; Boster, Wuhan, China; 1:10,000), and Peroxidase-goat Anti-mouse IgG (BA1051; Boster, Wuhan, China; 1:10,000).

The allograft kidney was isolated and lysed with radioimmune precipitation assay buffer (Beyotime, Shanghai, China) containing 1% NP40, 0.1% SDS, 100 µg mL⁻¹ PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail for 10 min, followed by filtered through a 45 µm strainer in the storage buffer (2% FBS in PBS). Then, the graft grinding supernatants were gathered after centrifugation at 13 000 × g for 20 min at 4 °C.

Total cellular or tissue proteins are extracted by using radioimmune precipitation assay buffer from Beyotime Biotechnology (China). Then 30 μg of proteins are run on a 12% SDS-polyacrylamide gel and examined by using the following antibodies: goat anti-PPARγ (Santa Cruz, catalog number sc-1984X), mouse anti-Egr-1 (Santa Cruz, catalog number sc-515830), and mouse anti-GAPDH (Proteintech, catalog number 60004-1-Ig).

Total cellular protein was extracted from cells using ice‐cold radioimmune precipitation assay buffer (Beyotime, Shanghai, China), and the concentration of protein was evaluated by the BCA protein assay kit (Beyotime). Twenty micrograms of protein was separated by 10% SDS/PAGE and then transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies against E‐cadherin, N‐cadherin and glyceraldehyde‐3 phosphate dehydrogenase overnight at 4 °C, followed by incubation with a secondary, horseradish peroxidase‐conjugated antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Then, these protein bands were measured using an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc, Basingstoke, United Kingdom). Glyceraldehyde‐3 phosphate dehydrogenase was used as an internal control.