Appropriate imaging system

Human gingival fibroblasts were starved in serum-free medium overnight before being stimulated for 30 min with salivary pellet that had been washed four times. Whole human saliva, tumor necrosis factor alpha (TNF-α, 10 ng/mL) and Interleukin (IL)-1 (10 ng/mL) served as positive controls, and serum-free medium as the negative control. Cell extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT). Primary antibody binding was accomplished with phospho-NF-kB p65 and β-actin antibodies (Cell Signaling Technology, Danvers, MA). Secondary antibody bindings were detected by near-infrared absorbing dyes with the appropriate imaging system (LI-COR Biosciences, Lincoln, NE).

Cell extracts containing SDS buffer and protease inhibitors (PhosSTOP with cOmplete; Sigma, St. Louis, MO, USA) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT, USA). Membranes were blocked and the binding of the first antibody (rabbit anti-pSmad3 Ser423/425, 1:500, Abcam, ab52903, Cambridge, UK), and actin (Santa Cruz Biotechnology, SCBT, Santa Cruz, CA, USA) was detected with the appropriate secondary antibody directly labeled with near-infrared dyes (LI-COR Biosciences, Lincoln, NE, USA) and visualized with the appropriate imaging system (LI-COR Biosciences). Acquired images were not processed.

Macrophages were serum-starved overnight and then treated overnight with CAPE as indicated. Cell extracts containing SDS buffer and protease inhibitors (PhosSTOP with cOmplete; Sigma, St. Louis, MO, USA) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT, USA). The membranes were blocked, and binding of the primary antibody (HO1 polyclonal antibody, Enzo Life Sciences, Farmingdale, NY, USA) was detected with an appropriate secondary antibody directly labelled with near-infrared dyes (LI-COR Biosciences, Lincoln, NE, USA) and visualised with an appropriate imaging system (LI-COR). The acquired images were not processed.