Odyssey western blot analysis system

Cells were harvested, washed in PBS (0.1 M, pH 7.4), centrifuged, and resuspended in cell lysis solution containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100 and several protein inhibitors such as sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃PO₄ and leupeptin (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration of each extract was determined by the bicinchoninic acid (BCA) assay [38 ]. Western blot was performed according to the procedure of Towbin et al. [39 (link)] with some modification. Cell extracts (60 μg protein/lane) were separated by electrophoresis on 12% SDS-polyacrylamide gels. Proteins were subsequently transferred to a nitrocellulose membrane, which was then incubated with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature. Afterward, the membranes were incubated with the primary antibodies (rabbit monoclonal anti-cleaved caspase-3 (1:100) and anti-β-actin (1:100)) overnight at 4 °C. After washing with TBST, membranes were then incubated with FITC-labeled secondary antibodies (Beyotime Institute of Biotechnology, Haimen, China), and the signal was read with an Odyssey^® Western Blot Analysis system (Li-COR Biosciences, Lincoln, NE, USA). The signal intensity of primary antibody binding was quantitatively analyzed with Sigma Scan Pro 5 (Systat Software Inc., San Jose, CA, USA)

Lung was rinsed with saline to remove blood. The lung was then homogenized and centrifuged at 10,000 rpm for 5 min. Thereafter, the supernatant was collected and protein concentrations were determined with the BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Proteins were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel. After electrophoresis, proteins were immediately transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) at 30 mA for 2 h. The membranes were blocked with 5% nonfat dry milk in TBS/Tween (25 mM Tris–HCl, 0.14 M NaCl, 2% Tween 20) at 4°C overnight. Membranes were incubated with primary antibody for one h and the antibody dilutions were as follows: anti-TGF-β1, 1:1000; anti-Smad7, 1:500; anti-p-Smad2/3, 1:500; anti-Collagen I, 1:1000. Washing between and after antibody incubation steps was performed three times for 10 min each with TBS/Tween buffer. Then, the membrane was incubated with fluorescent secondary antibody and scanned with Odyssey^® Western Blot Analysis system (LI-COR, Lincoln, NE, USA). The signal intensity of primary antibody binding was quantitatively analyzed with Sigma Scan Pro 5 and was normalized to the loading control β-actin.