Chemidoc it system

The human EGFR phosphorylation antibody array was obtained from RayBiotech (Peachtree Corners, GA, USA) and performed according to manufacturer’s instructions. The array is a dot-blot-based assay specifically designed to detect 17 different phosphorylation sites of the EGFR family, which includes four family members: EGFR, ErbB2, ErbB3, and ErbB4. Briefly, cell lysates obtained from Raji stimulated or not with refp17, EGF, NHL-a101 and NHL-a102 vp17s (0.1 μg/ml) for 5 min were added to antibody array membranes. Then, the membranes were washed and incubated with a cocktail of biotin-conjugated anti-EGFR. After incubation with HRP-streptavidin, the signals were visualized by chemiluminescence. Finally, the density of the immunoreactive dots was acquired by ChemiDoc-it System (Bio-Rad, Hercules, CA, USA) and protein phosphorylation and expression level was analyzed using Gel-Pro Analyzer 6.0 software (Media Cybernetics, Houston, TX, USA).

The quantity of IP-10-expressing hepatocytes was assessed by Western blot analysis. To this end, the isolated hepatocytes were counted and equal cell numbers were re-suspended in lysis buffer containing protease inhibitor cocktail (3 µl/ml, Sigma) and calyculin A (100 nM, Cell Signaling) followed by 10 min incubation on ice with repeated vortexing. Protein sample buffer (4x concentrated) was subsequently added (40% glycerol; 240 mM Tris; HCl pH 6.8; 8% SDS; 0.04% bromophenol blue; 7% β-mercaptoethanol) and the samples were denatured at 99 °C for 5 min. Samples were stored at −20 °C and denatured again at 99 °C for 5 min upon anew addition of 7% β-mercaptoethanol. The samples were separated using a 15% SDS-polyacrylamide gel followed by electroblotting onto a nitrocellulose membrane. Membranes were incubated first with the primary antibodies against IP-10 or α-tubulin (goat anti mouse IP-10, R&D Systems; rabbit anti mouse α-tubulin, Abcam) at 4 °C overnight and second with the respective horseradish-peroxidase-conjugated antibodies. The enzymatic reaction was detected using the ECL System (Santa Cruz Biotechnology) and the images were acquired by the ChemiDoc-it System (Bio-Rad). The relative concentration of IP-10 (ratio of volume under the integral of IP-10 and α-tubulin) was assessed using Image Lab Software (5.2.1 Bio-Rad).