Equal amounts of proteins mixed with NuPage loading buffer were loaded GSK3326595 datasheet on a 12% Bris-Tris Gels (Invitrogen) after being
denatured. After blockage the membrane was incubated using primary antibodies, which were added against ADAM17 (Abcam), HIF-1α (Cell VX-809 chemical structure Signaling), Sp1 (Santa Cruz) or β-Actin (Abcam) in PBS-T containing 5% milk overnight at 4°C. Subsequently, the membrane was washed with PBS-T for 45 minutes at room temperature, followed by incubation with the secondary antibodies for 1 hr and 30 min, still at room temperature. The immunoblots were detected, following a second washing, using the SuperSignal West Pico Chemiluminescent Substrate kit (Pierce). The internal control for Western blots was β-Actin. VEGFR inhibitor Alpha-secretase assay After U87 tumor cells were harvested, lysis buffer was added to the tubes. Proteins were sonicated five times for 10 sec each time. A BCA protein assay (Thermo Scientific) was done in order to find the protein concentration of each sample. A total volume of 200 μl proteins with buffers were added to the alpha-secretase specific APP (amyloid precursor protein) peptide. Two wells were used as control, where only buffer was added to them. Fluorescence was read using a Fusion multiplate reader (Packard Bioscience).
In vitro invasion assay Matrigel invasion assay (BD Biosciences) was employed to test cell invasion. The membrane was soaked in DMEM low glucose and incubated for one hour at 37°C. The bottom well contained high glucose DMEM containing serum. Cells were added to the upper well and incubated for 24 hours at 37°C with 5% CO2. After incubation, Cell Tracker (Invitrogen) dye was added to the wells for 20 minutes. Cells were fixed with 4% paraformaldehyde; the membrane was removed, and then transferred to slides for analysis. In vitro wound-repair assay This assay was used to assess cell migration. In a 24-well plate, U87 tumor Sulfite dehydrogenase cells were added to high glucose DMEM media, and incubated for two hours in order to create a monolayer of cells. A scratch was made in the middle
of the well with a p200 pipette tip. The debris was washed away and new media added to the wells. Under the microscope, the cells were imaged and the initial area of the scratch for the field of view was determined by multiplying the length by the average width of the area devoid of cells. The plate was incubated at 37°C for 12 hours, after which the same field of view was imaged and the area devoid of cells was recalculated by the same method. The final area of the scratch wound was divided by the initial area, giving us the % of the initial area covered by migrating cells over the 12 hours culture period. siRNA transfection A stable transfection of Sp1 siRNA was carried out using Lipofectamine 2000 transfection reagent (Invitrogen). The transfection reagent and the siRNA were diluted in 100 μL DMEM without serum or antibiotic.