FIG. 1.

(A) Effect of bortezomib treatment on HIF-1α and CAIX expression. Cells were seeded at 40,000/cm², allowed to attach overnight, pretreated with bortezomib for 30 min, and exposed to 21% (normoxia) or 0.5% O₂ for 24 h in the presence of bortezomib. Total protein lysates (40 μg) were tested for HIF-1α, CAIX, and β-actin by Western blotting. (B) Effect of bortezomib treatment on VEGF expression. VEGF, secreted by cells treated as described for panel A, was assayed with a VEGF sandwich enzyme-linked immunosorbent assay kit. VEGF levels are expressed as pg protein/mg of total protein, and each of the bars represents the mean value (±SD) from at least three individual experiments. (C) Effect of bortezomib treatment on the mobility of the Gal4-HIF-1α 529-826 fusion protein in MCF-7 cells. Cells were transfected with the Gal4-HIF-1α 529-826 construct, and 24 h later, they were treated as described for panel A and analyzed by Western blotting with anti-Gal4 DBD Ab.