Ras 3000 image analysis system

Total protein was isolated from the lungs by homogenization in radioimmunoprecipitation assay cell lysis buffer containing a mixture of protease inhibitor and phosphatase inhibitor (GenDEPOT, Barker, TX, USA), followed by centrifugation 13,000 rpm for 30 minutes at 4°C. Protein concentration was quantified using the BCA assay. The equal amount of sample protein (70 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). The membranes were blocked with 5% skim milk (Difco/Becton Dickinson, Atlanta, GA, USA) for 1 hour at room temperature, followed by incubation with a 1:100 dilution of anti-IRF5 (rabbit α-IRF5, Santa Cruz Biotechnology) in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 overnight at 4°C. After incubation, the blot was washed and incubated with a 1:10,000 dilution of anti-rabbit-immunoglobulin G, horseradish peroxidase-linked secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 hour at room temperature. After washing, the protein bands were visualized by enhanced chemiluminescence using a RAS 3000 Image Analysis System (Fujifilm, Tokyo, Japan).

Cells were seeded in 35 mm culture dishes (2×10³ cells/dish) and allowed to grow for 7 to 10 days in the presence of and/or absence of EGCG to form colonies. Colonies of more than 50 cells were visualized by crystal violet (in 60% methanol, Junsei Chemical, Japan) staining and images were taken by RAS 3000 Image Analysis System (Fuji Film, Japan).