Irdye 800 labeled secondary antibodies

Equal amounts of cell lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Then, membranes were stained with indicated primary antibodies, as follows: mouse anti-human TRX1 (1:500, BD Biosciences, 559969), rabbit anti-human GLRX1 (1:1000, R&D Systems, AF3399), rabbit anti-human actin (1:1000, Sigma, A2066), or mouse anti-human GAPDH (1:2000, Ambion, 4300), followed by staining with IRDye 680 or IRDye 800-labeled secondary antibodies (1:10,000, LI-COR Biosciences). Membranes were then scanned using a LI-COR Odyssey scanner (LI-COR Biosciences) (18 (link)).

16-HBE cells were treated with ARB, 3-hydroxyflavone, (+)-catechin, and (−)-epicatechin at indicated concentrations (12.5, 25, 50, 100, 200 μM) or treated with ARB at different incubation times (1, 2, 4, 6, 8 h) at 37°C for 4 h, the relative protein levels of STs were detected by using Western blot analysis. In brief, cells were washed with cold PBS for three times and lysed in RIPA buffer (containing protease/phosphatase inhibitors). After the protein concentrations were determined by using BCA protein assay (Pierce, Rockford, IL, USA), the absorbance of each sample was measured at 595 nm with a microplate reader (BioTek, Winooski, USA), then equal amounts of proteins (80 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on a nitrocellulose membrane. The nitrocellulose membrane was incubated with specific primary antibodies at 4°C overnight, followed by washing with PBS-T for three times. Then it was incubated with IRDye 800-labeled secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The detection was performed by the Odyssey Infrared Imaging System (LI-COR Biosciences).