Mouse Anti-VEGFA Recombinant Antibody (clone 2C3) (CAT#: HPAB-0060-YJ)

Figure 1 Anti‐VEGF‐A antibody 2C3 specifically recognizes VEGF‐A and decreases lymphatic vessel density in MDA‐MB‐231 xenografts.

a-Frozen sections of control and 2C3‐treated MDA‐MB‐231 tumors were double‐stained with antibodies against a pan‐endothelial marker CD31 (red color) and a marker of lymphatic vessels LYVE‐1 (green color). Note the dramatic reduction in vessel densities of both types on a representative section from a 2C3‐treated tumor. Magnification: ×200. b – Vessel densities were determined from double‐stained control (n = 33) and 2C3‐treated tumors (n = 36). Blood vessel density is presented as mean number of CD31‐positive structures per mm2 ± SD. Lymphatic vessel density is presented as mean number of LYVE‐1 positive structures per 100 mm² ± SD. Statistical differences were assessed by unpaired Student's t‐test. c – The total number of lymphatic vessels per cross‐section was determined on control (n = 33, black bars) and 2C3‐treated (n = 36, white bars) slides derived from individual mice and graphed according to the range in number of vessels in each section. d – Purified human VEGF‐A, ‐C and ‐D were immobilized on plastic and probed with the mouse monoclonal anti‐VEGF‐A antibody 2C3, rabbit polyclonal anti‐VEGF‐C antibody, or rabbit polyclonal anti‐VEGF‐D antibody. Primary antibodies were detected with appropriate HRP‐conjugated secondary antibodies and absorbance measured at 490 nm.

Whitehurst, B., Flister, M. J., Bagaitkar, J., Volk, L., Bivens, C. M., Pickett, B., ... & Ran, S. (2007). Anti‐VEGF‐A therapy reduces lymphatic vessel density and expression of VEGFR‐3 in an orthotopic breast tumor model. International Journal of Cancer, 121(10), 2181-2191.

Figure 2 Effect of anti‐VEGF‐A treatment on macrophage infiltration.

a, b-Representative images of sections from control (a) and 2C3‐treated (b) tumor stained with anti‐mouse CD11b antibody. C-The mean number of CD11b‐positive cells per high power field (×400) in control (n = 12) and 2C3 treated (n = 8) tumors ± SE.

Whitehurst, B., Flister, M. J., Bagaitkar, J., Volk, L., Bivens, C. M., Pickett, B., ... & Ran, S. (2007). Anti‐VEGF‐A therapy reduces lymphatic vessel density and expression of VEGFR‐3 in an orthotopic breast tumor model. International Journal of Cancer, 121(10), 2181-2191.

Figure 3 Anti‐VEGF‐A treatment does not affect VEGF‐C expression in MDA‐MB‐231 tumors in vivo.

a- Total RNA was extracted from snap frozen tumors and analyzed by endpoint RT‐PCR for the presence of VEGF‐C transcripts. PCR products from 3 representative control and 2C3‐treated tumors are shown. b – Densitometric ratios were calculated by normalizing VEGF‐C intensity to β‐actin intensity and are presented relative to control expression. Bars represent mean from 3 experiments. c-Protein lysates from 2C3‐treated and control tumors were analyzed for VEGF‐C expression via Western blot. Immunodetection of VEGF‐C and actin in representative tumors from each group is shown. d, e-Frozen tumor sections of control and 2C3‐treated tumors (d and e, respectively) were stained with rabbit anti‐VEGF‐C antibody recognizing both human and mouse VEGF‐C. Representative images from each group are shown. Similar expression of VEGF‐C protein was observed in all tumors regardless of treatment. Magnification is ×400.

Whitehurst, B., Flister, M. J., Bagaitkar, J., Volk, L., Bivens, C. M., Pickett, B., ... & Ran, S. (2007). Anti‐VEGF‐A therapy reduces lymphatic vessel density and expression of VEGFR‐3 in an orthotopic breast tumor model. International Journal of Cancer, 121(10), 2181-2191.

Figure 4 VEGF‐A and Ang‐2 modulate expression of VEGFR‐2 and VEGFR‐3 as well as induce Ang‐2‐dependent proliferation in lymphatic endothelial cells in vitro.

a-Total RNA was extracted from RLEC seeded in duplicate after 48 hr exposure to Ang‐2 or VEGF‐A165 (50 ng/ml each). VEGFR‐2 and VEGR‐3 expression was analyzed by qRT‐PCR, normalized by β‐actin and presented as percent increase in VEGFR‐2 and VEGFR‐3 expression of treated cells relative to control cells. The average values from 6 independent experiments ± SEM are presented. * denotes p < 0.002; NS – not significant. b – Ang‐2 or VEGF‐A₁₆₅ (0–100 ng/ml) were added to triplicate wells of RLEC in the absence or presence of soluble Tie‐2 (2 μg/ml). After 4 days, cells were trypsinazed and enumerated. The average cell numbers per well derived from 2 independent experiments are presented.

Whitehurst, B., Flister, M. J., Bagaitkar, J., Volk, L., Bivens, C. M., Pickett, B., ... & Ran, S. (2007). Anti‐VEGF‐A therapy reduces lymphatic vessel density and expression of VEGFR‐3 in an orthotopic breast tumor model. International Journal of Cancer, 121(10), 2181-2191.