Irdye 800cw conjugated antibody

Total mRNA was extracted from ventricles and primary cells using TRIZol reagent (Invitrogen), and cDNA was synthesized using oligo (dT) primers with the Transcriptor First Strand cDNA Synthesis Kit (Roche). Selected gene differences were confirmed by quantitative real-time PCR using SYBR green (Roche) and the results were normalized against GAPDH gene expression. The primers for real-time PCR are shown in Supplementary Table 7. Total protein and nuclear protein were extracted from ventricles and primary cells, respectively37 (link). The protein concentration was determined using the Pierce BCA Protein Assay kit (Pierce). Fifty micrograms of protein was used for SDS–PAGE (Invitrogen), and the proteins were transferred to a polyvinylidene fluoride membrane (Millipore), which were incubated with various primary antibodies overnight at 4 °C. After incubation with a secondary IRDye 800CW-conjugated antibody (Li-Cor Biosciences, at 1:10,000 dilution), signals were visualized with an Odyssey Imaging System (Li-Cor Biosciences). The specific protein expression levels were normalized to GAPDH for the total lysates or to Lamin B for the nuclear proteins on the same nitrocellulose membrane. Full gel scans are shown in Supplementary Fig. 9.

Extracts of cell lysate and tissue were analyzed by western blotting using the IRDye 800CW-conjugated antibody and LI-COR imaging system (LI-COR Biosciences, Lincoln, NE, USA) according to the manufacturer's instructions. Membranes were scanned and analyzed using an Odyssey IR scanner and Odyssey imaging software 3.0.

Renal tissues and NRK-52E cells were lysed with radioimmunoprecipitation assay lysis buffer (Biyuntian, Haimen, China) on ice and the lysates were centrifuged for 15 min (12,000 × g, 4°C). Protein concentrations were determined with a bicinchoninic acid protein assay kit (Sigma-Aldrich; Merck KGaA). Proteins (20 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (EMD Millipore; Billerica, MA, USA). Following blocking with 5% non-fat milk at room temperature for 2 h, the membranes were probed with the primary antibodies at 4°C overnight. The antibodies used were as follows: Anti-β-actin (1:400; sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA), anti-α-SMA (1:1,000; BM0002; Wuhan Boster Bioengineering Co., Ltd.), anti-E-cadherin (1:1,000; 610181; BD Biosciences). Membranes were subsequently incubated with a secondary IRDye^® 800CW-conjugated antibody (1:1,000; 925-32211; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The signals were visualized with an Odyssey Imaging System (Odyssey; LI-COR Biosciences, USA) and the target protein expression levels were normalized to those of β-actin. Protein levels were assessed using ImageJ 1.46 software (National Institutes of Health, Bethesda, MD, USA).