Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1
Figure 2
VEGF Receptor Expression in VEGF-Reduced MDA-MB-231 Cells
(A) MDA-MB-231 cells and HBMECs were treated with VEGF (30 ng/ml) to examine its effects on the phosphorylation (top blots) and expression (bottom blots) of VEGFR2. Total cell lysates were prepared and then subjected to immunoprecipitation (IP) and Western blotting (WB) using 4G10 or anti-VEGFR2 antibodies. The arrowhead indicates a nonspecific protein band.
(B) Lysates (5 mg) from MDA-MB-231 cells were immunoprecipitated by using anti-VEGFR1 or anti-VEGFR2 antibodies. The immune complexes were analyzed by Western blotting using anti-VEGFR2 antibodies. After stripping, the membrane was subjected to Western blotting using anti- VEGFR1 antibodies.
(C) Lysates (2 mg) from variously transfected MDA-MB-231 cells were subjected to immunoprecipitation (IP) and Western blot (WB) analyses for VEGF and NRP1 receptor expression, as indicated. Total protein lysates (0.4 mg) from HBMECs were used as a positive control.
(D) Total RNA (5 μg) from variously transfected MDA-MB-231 cells was subjected to RT-PCR analysis for VEGFR1 or VEGFR2 mRNA expression. GAPDH mRNA is shown as an internal control.