Imagequant tl program

Protein extracts from 28 days post-injection B16F10 tumors were prepared. Proteins (50 μg/lane) were separated by 12% SDS−PAGE and electroblotted. Blotted membranes were cut at the 35 kDa molecular weight. The lower part was incubated with rabbit anti-cleaved caspase-3 mAb and the upper part with mouse anti-β-tubulin mAb followed by incubation with goat anti-mouse or goat anti-rabbit IgG peroxidase conjugate. The blots were developed and densitometric data were calculated using the ImageQuantTL program (GE Healthcare Life Sciences, Chicago, IL, USA). The caspase-3 signal was normalized to β-tubulin and quantified using EZQuant-Gel software.

Cell lysates were harvested in NP-40 lysis buffer containing protease inhibitor cocktail (Roche Applied Bioscience) and quantitated using a Pierce bicinchoninic acid protein assay kit (Fisher Scientific). Equivalent amounts of protein were separated in Mini-Protean TGX precast 4 to 20% gels (Bio-Rad Laboratories, Hercules, CA, United States) and transferred to nitrocellulose membranes. Membranes were blocked in phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20 and incubated with primary antibody. The following antibodies were used: anti-S-tag (1:1000; Abcam, Cambridge, MA), anti-UBR5 (1:1000; Cell Signaling Technology, Danvers, MA, United States), anti-HBZ (1:1,000), anti-APH-2 (1:1,000), anti-FLAG clone M2 (1:1,000; Agilent Technologies, Santa Clara, CA, United States), anti-ubiquitin (1:250; Santa Cruz Biotechnology), anti-β-actin (1:5,000; Sigma–Aldrich), and anti-α-Tubulin (1:250; Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase-labeled goat anti-rabbit and goat anti-mouse immunoglobulin antibodies (1:5,000; Santa Cruz Biotechnology). The blots were developed using an ECL Western Blotting Substrate (Fisher Scientific). Images were taken using an Amersham Imager 600 imaging system (GE Healthcare Life Sciences), and densitometric data were calculated using the ImageQuant TL program (GE Healthcare Life Sciences).

Cells growth, UV-irradiation and DNA extraction was done as described in ‘Quantification of RDHs and PDs in the chromosomal DNA’. 10 μg of DNA was digested with HaeII in 200 μl reaction in the 1xCutSmart Buffer (NEB) at 37°C, followed by extractions with phenol, phenol/chloroform, chloroform and ethanol precipitation. DNA samples were split in half and run on two agarose gels. Treatment of the gels, processing of the membranes and western analysis were as described in ‘Quantification of RDHs and PDs in the chromosomal DNA’, the only difference being that DNA from both gels was transferred to the membranes by electric transfer. Western signal imaging was done with iBright CL1000 Imaging System (Invitrogen). Image analysis and quantification of the TIFF files was done with ImageQuant TL program (GE HealthCare Life Sciences).