Licor odyssey infrared image system

Protein from renal cortex and cultured renal cell lines was extracted using the radio immunoprecipitation assay (RIPA) lysis buffer. Western blot analysis was performed as pervious described 12 (link). Antibodies involved such as collagen-I (Col-I), α-smooth muscle actin (α-SMA), phospho-NF-kB/p65, phospho-Smad3, Smad3, Smad7 and β-actin were described as previous 14 (link), 15 (link). Then, IRDye800-conjugated secondary antibodies (Rockland Immunochemical, Gilbertsville, PA) were used as secondary antibodies. Signals were detected using the LiCor/Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE), followed by quantitative analysis using the Image J program.

The cell-expressed GST and the GST-N fusion proteins were separated by 12% SDS-PAGE and the protein samples were then transferred onto nitrocellulose filter membrane (PALL, New York, USA). The membranes were incubated with ascites fluid and anti-GST mAb as the primary antibody. After the membranes were rinsed with PBS, each membrane was treated with IRDye-700-conjugated goat anti-mouse IgG (LI-COR Biosciences, USA) as the secondary antibody. The proteins were visualized by scanning the membranes with the LI-COR Odyssey infrared image system (LI-COR Biotechnology, USA).

Protein was extracted from HK-2 cells and the kidney tissue of mice by radioimmunoprecipitation (RIPA)-Buffer (Beyotime, Jiangsu, China). A bicinchoninic acid (BCA) protein quantization kit (Beyotime, Jiangsu, China) was used to determine protein concentration. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were sealed with 5% milk for 2 h, incubated with KIM-1 (1:800), p-P65 (1:1000), RIPK1 (1:800), RIPK3 (1:800), cle-caspase-3 (1:1000), and β-actin antibodies (1:1000). This was incubated with IRDye 800-conjugated secondary antibody for 1.5 h at 25 °C. Finally, immunoreactive signals were detected with the LiCor/Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE) and analyzed by Image J software (NIH, Bethesda, MD, USA). The experiment was repeated three times, using β-actin as an internal control.

Proteins from kidney tissues and cultured cells were extracted with RIPA lysis buffer, and western blot analysis was performed as described previously [10 (link)]. After blocking the nonspecific binding with 5% BSA (1h, room temperature), membranes were then incubated with the primary antibody against phospho-Smad3 (Cell Signaling Technology), collagen I (Southern Biotech), Smad7, Smurf2, total Smad3 (Santa Cruz Biotechnology), α-SMA, glyceraldehyde 3-phosphate dehydrogenase (Chemicon, Temecula, CA) overnight at 4°C, followed by the IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA). Signals were captured using the LiCor/Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE) and the intensity of each band was quantified and analyzed by using the Image J software (NIH, Bethesda, MD, USA).

Total proteins of tumour or normal (the skin of the same mouse) tissues were extracted by chilled RIPA lysis buffer (Pierce) and then subjected to the western blotting analysis with primary antibodies against CD31, VEGF, MMP-2, MMP-9, MMP-13, CXCR4, NKp46, p-Smad3, Smad3 (all from Santa Cruz Biotechnology) and E4BP4 (Cell Signaling) in 1:1,000, followed by incubation with the corresponding IRDyeTM800-conjugated secondary antibodies (1:10,000, Rockland Immunochemicals). β-Actin was used as an internal control. Expression levels of the proteins were detected by using LiCor/Odyssey infrared image system (LI-COR; Biosciences), and the band intensities were quantified with the Image J software (version 1.48, NIH, Bethesda). Images have been cropped for presentation; their full size images are presented in Supplementary Figs 15–20.

Proteins from tumor tissue or cultured cells were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer and subjected to SDS-PAGE and then electro-transfer to nitrocellulose membranes. After blocking with 5% BSA/Tris-buffered saline (TBS) buffer, membranes were incubated with primary antibodies against mouse p-Smad3, Smad7, p-ALK5 (Abcam, MA, USA), MMP2, MT1-MMP, TIMP2 (Merck Millipore, MA, USA), MMP9, MMP13, β-actin (Santa Cruz Biotechnology, CA, USA), and NF-κB p50 (Cell Signaling, MA, USA) dissolved in 1% BSA overnight at 4°C, followed by incubating with IRDye 800-conjugated secondary antibody (Rockland Immunochemicals, PA, USA). Signals of target proteins were detected by Li-Cor/Odyssey infrared image system (LI-COR Biosciences, NE, USA) and then analyzed with ImageJ software (NIH, Bethesda, MD, USA).

Protein from kidney tissues and cultured cells was extracted with RIPA-Buffer (Beyotime, Jiangsu, China); BCA kit (Beyotime, Jiangsu, China) was used to measure concentration. Western blot was performed as described previously 19 (link), 21 (link). The membrane was incubated with 5% milk in PBS for two hours to block nonspecific binding, then incubated with appropriate antibody using rabbit anti-KIM-1, anti-P-p65/p65, anti-P-p53/p53, anti-cleaved casapase-3, anti-RIPK1, anti-RIPK3, anti-P-Smad3/Smad3 and mouse anti-GAPDH overnight at 4°C. Membrane was incubated with IRDye 800-conjugated secondary antibody for 2 hours at room temperature (Rockland immunochemicals). Images were captured with LiCor/Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE) and then analyzed by Image J software (NIH, Bethesda, MD, USA).