Proteome profiler mouse angiogenesis array

WT mice and db-/db- mice have been operated as described. 5 mm large areas of the tibia including the defect have been harvested and stored at −80°C until usage. Tissue has been mechanically homogenized and tissue fragments were collected in PBS supplemented with complete protease inhibitor cocktail (Roche). Non-operated WT and db-/db- tibiae served as controls. Two tibiae were pooled for analysis. Proteome profiler mouse angiogenesis array (R&D Systems, ARY015) was performed according to manufactures’ instructions. Equal total protein amounts were used for each sample. Protein concentration determination was carried out with BCA protein assay kit (Pierce) according to manufactures´ instructions and ELISA reader ELx800 (Biotek). For quantification, developed x-ray films were scanned, and pixel density of each spot of the array was determined with Image J. For data analysis, average background signal (negative control spots) was subtracted from duplicate spot signal intensity which was subsequently normalized to positive control spots and related to signals from WT mice.

To measure the secretion of angiogenesis-related proteins, conditioned media was collected from prostate cancer cells treated with SCF. RM1 or mPC3 cells were grown on 10 cm tissue culture dishes and incubated at 37°C until they reached 70-80% confluence. The cells were then washed with serum-free media and treated with 50 ng/mL murine recombinant SCF for 24 hours. The cell culture supernatant was collected and centrifuged at 300 g for 10 min to remove cell debris. The conditioned media was stored at -80°C until further use. The Proteome Profiler Mouse Angiogenesis Array (R&D Systems, RRID: AB_1655573) was used in accordance with the manufacturer’s protocol with 700 µL of thawed supernatant. The array was analyzed by densitometry using Bio-Rad ImageLab. Proteins were normalized and compared to cells grown without SCF to calculate fold-change.