Imager 600rgb

FaDu cells were seeded in 10-cm culture plates, starved 24 hours in serum-free medium and then, with and without AvidinOX conjugation, cultivated 48 hours in the presence or absence of bCet 5 μg/mL, with EGF induction the last 30 minutes (100 ng/mL). Whole cell lysates were then prepared and used for assessing the expression of various proteins involved in apoptosis and DNA repair, through the Human Apoptosis Array kit (R&D), according to manufacturer's instructions.
After exposition of membranes to X-ray film, pixel densities were collected through the Imager 600RGB (GE) and analyzed by image analysis software (ImageQuant TL Image Analysis Software v8.1, GE). Average background signal was subtracted from each spot and the normalized mean pixel densities plotted according to target family. Data were expressed as percentage with respect to values measured in control cells.

The following primary antibodies were used: rabbit polyclonal anti-LEDGF/p75 (Bethyl Laboratories; product code A300-848A), rabbit polyclonal anti-HRP2 (Novus Bio; product code NBP2-47438), affinity-purified polyclonal rabbit anti-MVV capsid/p24 antibody (Thermo Fisher Scientific, custom product; see Supplementary Fig. 12 for validation data), and horseradish peroxidase-conjugated rabbit monoclonal anti-β-actin antibody clone 13E5 (Cell Signaling Technology, product code 5125). Blots probed with anti-LEDGF/p75 (diluted 1:5000 in phosphate-buffered saline, supplemented with 0.05% Tween-20 (PBST)), anti-HRP2 (diluted 1:1000 in PBST), and anti-capsid/p24 antibodies (1:5000) were developed following incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (BioRad; product code 1706515; diluted 1:10,000 in PBST) or IRDye 800CW conjugated goat anti-rabbit IgG antibody (LI-COR; product code 925-32211; diluted 1:2000 in PBST) for detection by chemiluminescence, using Imager 600 RGB (GE Healthcare) following incubation with ECL prime reagent (GE Healthcare) or by fluorescence, using an Odyssey imager (LI-COR), respectively.

SKBR3 cells were seeded in 10-cm culture plates, starved 24 hours in serum-free medium and then, with and without AvidinOX conjugation, cultivated 24–72 hours with antibodies or medium alone. Whole protein extracts were then prepared, as previously described for Western blotting analysis, and used for assessing the expression of various apoptosis-related proteins and the content of phosphorylated kinases and their substrates, through the specific Human Apoptosis and Human Phospho-Kinase Proteome Profiler Array kits (R&D Systems), respectively, according to manufacturer's instructions. After exposition of membranes to X-ray film, pixel densities were collected through the Imager 600RGB (GE) and analyzed by image analysis software (ImageQuant TL Image Analysis Software v8.1, GE). Average background signal was subtracted from each spot and the normalized mean pixel densities plotted according to target family. Data were expressed as percentage with respect to values measured in control cells.