Streptavidin cy3

The obtained protein sample (50 ug) was brought to 75 ul with labeling buffer, treated with 3 ul of 10 ug/ul biotin/DMF solution, and incubated at RT for 1 h with mixing. Stop reagent (35 ul) was added, and the sample was incubated at RT for 30 min with mixing. The antibody microarray slide (Fullmoon Biosystems) was shaken (55 rpm) with 30 ml of blocking solution in a petri dish for 1 h at RT. After blocking, the slide was rinsed with Milli-Q grade water. The labeled sample was mixed with 6 ml of coupling solution, and the blocked array slide was shaken with the coupling mixture (60 rpm) for 2 h at RT in a coupling dish. The slide was then washed six times with 30 ml of washing solution in a petri dish with shaking (55 rpm) for 5 minutes, and then rinsed with Milli-Q-grade water. For detection, 30 ul of 0.5 mg/ml Cy3-streptavidin (GE Healthcare, Chalfont St. Giles, UK) was mixed with 30 ml of detection buffer and shaken (55 rpm) with the coupled array slide for 20 minutes at RT. The slide was washed six times with 30 ml of washing solution in a petri dish (55 rpm, 5 minutes each), and rinsed with Milli-Q-grade water.

Antibody arrays were purchased from Full Moon BioSystems (PMK185 and PEG214). Biotinylation of the proteins and conjugation and detection by Cy3-streptavidin (PA43001; GE Healthcare Life Science, Little Chalfont, UK) were performed using an antibody array assay kit (Full Moon BioSystems) according to the manufacturer’s instructions. Protein samples (60 μg) were used in the antibody array assay. To calculate the STR using Eq. (5), the detection assay was performed six times independently following the addition of EGF to the cultures (50 ng/mL using the PEG214 array and 100 ng/mL using the PMK185 array). The mean fluorescent intensity (± standard deviation) was utilized to calculate the transduction characteristics. The antibody arrays were scanned using a SureScan Microarray Scanner (G2565CA Microarray Scanner System; Agilent Technologies, Santa Clara, CA, USA), after which the acquired image data were analyzed. The signal intensity was normalized by dividing the result by the negative control values from the array. Each value for phosphorylated proteins was divided by the respective value for unphosphorylated proteins at 0, 15, 30, 45, 60, 120, and 180 min. Finally, the results were divided by the value at 0 min to calculate the increase in phosphorylated molecules. The coefficient of variation for six replicates was < 0.1^{33 (link)}.

Phospho Explorer antibody arrays were used to assess differences in serum protein profiles between the three groups. Protein array assays were performed according to the manufacturer's instructions. Briefly, whole serum was depleted of albumin (see below), diluted in labeling buffer (1:15), and mixed with Biotin/DMF labeling solution. Biotinylated proteins from each sample were then incubated on individual antibody-coated slides, followed by fluorescence labeling with Cy3-streptavidin (GE Healthcare). The slides were scanned on an Axon GenePix Array Scanner (Molecular Devices) to detect bound proteins based on fluorescence intensity. Fluorescence intensity of each protein array was normalized and analyzed using GeneSpring software v. 12.5 (Agilent, Santa Clara, CA, USA).

The metaphase chromosomes were prepared from infected T cells on day 14 post-infection (dpi) and analyzed for the presence of the MDV genome by FISH [24 (link),31 (link)]. Briefly, MDV genomes were detected using a set of PCR-based MDV probes that were generated using the Biotin PCR Labeling Kit (PromoCell, Heidelberg, Germany) (for primers, see Table 1). Virus genomes were visualized using Cy3 Streptavidin (1:1000; GE Healthcare, PA43001; Munich, Germany), metaphase FISH images were taken using an Axio Imager M1 system and the AxioVision software (Carl Zeiss, Inc.; Oberkochen, Germany) and analyzed with ImageJ (https://imagej.nih.gov/ij/, accessed on 28 November 2021). Appropriate positive and negative controls were included (Figure S2).

The in situ hybridization protocol was adapted from Reineke et al.^{56 (link)} Cells were fixed with 2% formaldehyde in PBS for 1 min at room temperature and permeabilized with ice-cold methanol for 10 min at −20 °C. The following 5′-biotinylated DNA probe was used to detect 28S rRNA: 5′-cggcgctgccgtatcgttccgcctgggc gggattctgacttagaggcgttc-3′. The probe was hybridized in hybridization buffer (50% formamide, 2 × SSC, 10% dextran sulfate, 0.2% BSA, 5 mM DTT) at a final concentration of 1 μg/ml for 24 h at 43 °C in a humidified chamber and detected by subsequent incubation with Cy3 streptavidin (GE Healthcare Amersham, Milan, Italy) in 4 × SSC plus 0.1% Triton X-100 for 1 h at room temperature. Blocking and incubation with primary and secondary antibodies were next performed as described above.

The glass-slide-based phospho-explorer antibody array was utilised according to the manufacturer’s instructions (Full Moon BioSystems Inc.). ZR-75-1 and MDA-MB-468 cells treated with 50 μM AG-205 and PGRMC1 siRNA transfected were harvested using protein extraction buffer (Full Moon BioSystems Inc.) diluted in labelling buffer and mixed with Biotin/DMF labelling solution. Biotinylated proteins were incubated on individual antibody-coated slides, and were labelled with Cy3-streptavidin (GE Healthcare). To detect fluorescent intensity, the slides were scanned using an Axon GenePix Array Scanner (Molecular Devices). The intensity values obtained were normalised relative to internal controls and analysed.