Biomax mr and xar film

Proteome profiler antibody based Angiogenesis array (R & D systems; Cat. No. ARY007) was used according to manufacturer’s instructions to detect the relative levels of expression of angiogenesis related proteins in control and Bazedoxifene treated cells. After blocking the membrane, 300μg of protein from untreated or Bazedoxifene (2.25μM) treated cells was added and incubated overnight at 40°C. Next day, the membrane was washed and streptavidin-HRP was added for 30 minutes. Immunoreactive signals were visualized using Super Signal Chemiluminiscence substrate (Pierce) and Biomax MR and XAR film (Eastman Kodak Co.). Array data on developed X-ray film was quantified by scanning the film using Biorad Molecular Image Gel DocTM XR+ and analyze the data using Image LabTM software.

Proteome Profiler Human Angiogenesis Array Kits (R&D Systems, Minneapolis, MN) were used per the manufacturer’s instructions to detect the relative expression levels of 55 angiogenesis-related proteins in conditioned media-treated HUVECs and MV-treated mice bearing intracranial D283med-luc tumors. For HUVEC studies, whole cell lysate was made from HUVEC cells treated with 100 μg/ml MV-GFP or MV-hE:A conditioned media for 24 hours. After blocking the membranes, 300 μg of protein from the samples were added and incubated overnight at 4°C. The membranes were washed the next day and streptavidin-HRP was added or 30 minutes. Immunoreactive signals were visualized using Super Signal Chemiluminiscence substrate (Pierce) and Biomax MR and XAR film (Eastman Kodak Co.). Array data on developed X-ray film was quantified by scanning the film using Biorad Molecular Image Gel Doc™ XR + and analyzed using Image Lab™ software. Arrays for the in vivo studies were conducted in a similar fashion, using 300 μg lysate derived from excised D283med-luc tumors three days following MV treatment. Two tumors were analyzed for each treatment group.