2.17. Angiogenesis Array

The human angiogenesis array (Proteome Profiler™ Array; R&D Systems.) was used to assess the relative expression of 55 angiogenic-related proteins (array map provided in Figure S1b) in cellular extracts of siNS- and siEPHA2-transfected MKN74 cells and in siNS-transfected MKN74 cells infected for 24 h with H. pylori 60190, which was used as a reference for the angiogenic response. The array membranes were probed with combined cellular extracts from 3 independent experiments with a total protein content of 250 µg per condition, according to the manufacturer’s instructions. Enhanced chemiluminescence was used to detect protein binding to the antibody array, followed by exposure to an X-ray film. The signal intensity of each antigen-specific antibody spot was quantified using Quantity One^® image analysis software (BioRad). For comparison of the relative expression of proteins between siNS- and siEPHA2-transfected cells in uninfected (U) and H. pylori-infected (I) conditions across the different arrays, the mean pixel density of the duplicated spots for each protein after subtraction of the mean pixel density of the negative control spots of the respective array was normalized for the mean pixel density of the positive control spots on the reference array (siNS_U), according to the following formula: Normalized signal intensity for protein X in array A = Mean signal density for protein X in array A * (mean signal density of positive control spots on reference array/mean signal density of positive control spots on array A). Heat map analysis using the normalized data was performed in the R software [49] using the “gplots” package. IL-8 quantification by ELISA in conditioned media from 6 independent experiments was used as a validation hit of the antibody array.