Odyssey infrared imaging system scanner

Cells were with NP-40 (Thermo Scientific) plus Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentration was measured using the BCA Protein Assay (Thermo). Western blots were performed using NuPAGE 4–12% Bis-Tris Protein Gels (Invitrogen). For gel electrophoresis, the iBlot 2 Dry Blotting System (Invitrogen) was used with MES buffer (Invitrogen) and transferred onto nitrocellulose transfer membrane. Membranes were incubated with Odyssey blocking buffer (Li-Cor) prior to incubation with primary antibodies overnight at 4°C. Goat anti-rabbit IgG (H+L) 800 CW or goat anti-mouse (H+L) 680RD was applied for 45 minutes at room temperature (1:15000, LI-COR) before washing with PBS with Tween 20. An Odyssey Infrared Imaging System Scanner was used to generate immunoblot images and the LI-COR Odyssey scanner and software (LI-COR Biosciences) were utilized for band quantification. The antibodies used were specific for AR (5153; Cell Signaling; 1:2000), ETV1 (PA5-41484; ThermoFisher Scientific; 1:1000), IGF1 (PA5-27207; ThermoFisher Scientific; 1:1000), VEGF (PA5-16754; ThermoFisher Scientific; 1:200), and β-actin (3700; Cell Signaling; 1:2000).

Before the incubation of cellular extracts, each pad of the microarray was blocked with 5% BSA (w/v) in Tris buffered saline +0.1% Tween 20 (TBS-T) for 2 h at 4 °C with gentle shaking. After three washing steps with TBS-T at RT, each pad was incubated for 2 h at 4 °C with 80 µL of samples containing total protein extracts (with a total protein concentration of 1 mg/ml). Unbound proteins were removed by washing the array for 3 × 5 min with TBS-T.
Incubation with anti-RPA2 or anti-DNA-PKcs biotinylated antibody was performed for 2 h at 4 °C. The slides were washed again and were then incubated for 1 h in the dark with streptavidin-conjugated QDs 705 (4 µg/ml, Thermofisher, ref Q10151MP). The final washing step (3 × 10 min) with TBS-T was done before detecting the fluorescence signal with an Odyssey infrared imaging system scanner at a resolution of 21 µm (LI-COR Biosciences, Lincoln, NE, USA). Light excitation was performed at 685 nm.

The MIAA assay allows testing for panels of commercially available proteins, antigens, or/and lysates from EBV, herpes simplex virus 1 (HSV-1), HSV-2, cytomegalovirus (CMV), varicella zoster virus (VZV), HCV, Helicobacter pylori (H. pylori), Toxoplasma gondii (T. gondii), and Borrelia burgdorferi (B. burgdorferi) (12 (link)–15 (link), 21 (link)). For incubation on MIAA arrays, Ig concentrations were adjusted to 400 μg/ml (serum) or 50–200 μg/ml (purified monoclonal IgAs) in 80 μl. After washing, MIAA slides were incubated with Dylight^TM 680-labeled goat anti-human IgA Fc antibody (1:2,500; 0.4 μg/ml; Immuno Reagents, Raleigh, NC, USA). Fluorescence signals were detected with the Odyssey infrared imaging system scanner at 21-μm resolution (LI-COR Biosciences, Lincoln, NE, USA) and quantified using the GenePix® Pro 4 Microarray Acquisition and Analysis Software (Molecular Devices, Sunnyvale, CA, USA) (12 (link)–15 (link), 21 (link)). Five fluorescence thresholds of specific positivity were determined using positive and negative controls: 500, for HCV, H. pylori, T. gondii; 1,000, for HSV-1 and HSV-2; 1,200, for CMV; 1,400, for EBV and VZV; and 1,800 for B. burgdorferi. Fluorescent signals below the thresholds were considered negative (12 (link), 21 (link)).