Blocking solution

Cell lysates obtained from HPMEC cells (treated with recombinant human VEGFA and recombinant human CCL28, with PBS as a control) were applied to the VEGFR and GPCR-MAPK Pathway Phosphorylation Antibody Array (Full Moon BioSystems, USA), containing 185 and 193 antibodies, respectively. Each of the antibodies has 6 replicates that are printed on standard-size coated glass microscope slides. In brief, The Antibody Array was first blocked with blocking solution (Full Moon BioSystems, USA) for 30 minutes at room temperature, followed by incubation with the biotin-labeled cell lysates at 4 °C overnight. After washing 3 times, the conjugated labeled proteins were detected using Cy3-conjugated streptavidin. For each antibody, phosphorylation ratio was computed as the equation: phosphorylation ratio = (phopho_expeiment/unphospho_experiment)/(phopho_control/unphospho_control). The results of the Phospho-antibody array were further confirmed by Western Blot assay.

The anti-phosphoprotein microarray PGP193, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 193 antibodies. Each of the antibodies has six replicates that are printed on coated glass microscope slides, along with multiple positive and negative controls. In brief, cell lysates obtained from HEC-1B/mock and HEC-1B/shAMF-1 were biotinylated with the Antibody Array Assay Kit (Full Moon Biosystems, Inc.). The antibody microarray slides were first blocked in a blocking solution (Full Moon Biosystems, Inc.) and dried with compressed nitrogen. The slides were then incubated with the biotin-labeled cell lysates (∼100 μg protein) in coupling solution at room temperature for 2 h and rinsed extensively with Milli-Q grade water before detection of bound biotinylated proteins using Cy3-conjugated streptavidin. The slides were scanned on a GenePix 4000 scanner and the images were analyzed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). The fluorescence signal for each antibody was obtained from the fluorescence intensity of this antibody spot. A ratio computation was used to assess the extent of protein phosphorylation. For each antibody that has phosphorylated and matching unphophorylated values in both the control data and experiment data are represented as “phospho” and “unphospho”.

Cell cycle-related protein profile was determined using the Cell Cycle Control Phospho Antibody Array (95 site- and phospho-specific antibodies, Fullmoon Biosystems, Sunnyvale, CA, USA) according to the manufacturer´s guidelines. Briefly, slides were treated with blocking solution (Fullmoon Biosystems) for 30 min at room temperature and incubated with 75 μg of biotin-labelled first trimester placental protein lysates overnight at 4 °C. After washing, conjugated proteins were detected using Cy3-conjugated streptavidin. Image analysis was performed using GenePix Pro 7.0 software (Molecular Devices, San Jose, CA, USA). After local background subtraction, data were normalized on the median intensity value of all antibodies on each array (excluding empty spots and negative/positive markers). Only those signals exceeding the background intensity by two-fold were considered.