Anti-VEGF/VEGFA Antibody Picoband®

Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells. Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, MMP9, E-cadherin, N-cadherin, snail, slug, and GAPDH were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs respective control).
Index in PubMed under a CC BY license. PMID: 26811493

Depletion of PLCγ1 suppresses growth and metastasis of gastric adenocarcinoma in a nude mouse tumor xenograft model. ( A ) Volume and weight of tumor samples from nude mice. ( B ) The protein levels of PCNA, cleaved-PARP, PARP, and Bcl-2 in the tumor samples were detected by Immunohistochemistry (Magnificationx100, x400) and Western blotting analyses as described in Materials and Methods. ( C ) The levels of MMP2 and MMP9 in the tumor samples were detected by Immunohistochemistry analysis as described in Materials and Methods (Magnificationx100, x400). The protein and mRNA levels of MMP2, MMP9, E-cadherin(CDH1), N-cadherin(CDH2), snail(SNAIL), and slug(SLUG) in the tumor samples were detected by Western Blotting and Real-time PCR analyses as described in Materials and Methods. ( D ) The protein levels of VEGF and CD34 and the mRNA level of VEGF in tumor samples were detected by Immunohistochemistry and Real-time PCR analysis as described in Materials and Methods. The number of microvessels was accounted under OLYPUS x41 microscope (Magnification x100, x400). ( E ) The lymphoid follicles in inguinal lymph nodes of nude mice were observed under OLYPUS x41microscope, and the protein levels of CD44 and GFP in inguinal lymph nodes of nude mice was detected by Immunohistochemistry analysis as described in Materials and Methods (Magnificationx40, x400). Data are reported as means ± S.D. of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs respective control).
Index in PubMed under a CC BY license. PMID: 26811493

Western blot analysis of VEGF/VEGFA using anti-VEGF/VEGFA antibody (PB9071).
Electrophoresis was performed on a 12% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: recombinant human VEGF/VEGFA protein 20ng,
Lane 2: recombinant human VEGF/VEGFA protein 10ng,
Lane 3: human U87 whole cell lysates,
Lane 4: human K562 whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat C6 whole cell lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse Neuro-2a whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-VEGF/VEGFA antigen affinity purified polyclonal antibody (PB9071) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054) at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for VEGF/VEGFA at approximately 18, 27 kDa. The expected band size for VEGF/VEGFA is at 27 kDa.

IHC analysis of VEGF/VEGFA using anti-VEGF/VEGFA antibody (PB9071).
VEGF/VEGFA was detected in a paraffin-embedded section of human ovarian serous adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-VEGF/VEGFA Antibody (PB9071) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of VEGF/VEGFA using anti-VEGF/VEGFA antibody (PB9071).
VEGF/VEGFA was detected in a paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-VEGF/VEGFA Antibody (PB9071) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Flow Cytometry analysis of K562 cells using anti-VEGF/VEGFA antibody (PB9071).
Overlay histogram showing K562 cells stained with PB9071 (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-VEGF/VEGFA Antibody (PB9071, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.